The present invention provides, among other things, chimeric dna polymerases containing heterologous domains having sequences derived from at least two dna polymerases that have at least one distinct functional characteristics (e.g., elongation rate, processivity, error rate or fidelity, salt tolerance or resistance) and methods of making and using the same. In some embodiments, the present invention can combine desired functional characteristics (e.g., high processivity; high elongation rate; thermostability; resistance to salt, PCR additives (e.g., PCR enhancers) and other impurities; and high fidelity) of different dna polymerases in a chimeric polymerase.
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1. A dna polymerase of whose amino acid sequence shows at least 95% overall identity with SEQ ID NO:16, the dna polymerase characterized in that:
a) it shows a higher fidelity than a wildtype KOD polymerase; and
b) it shows higher salt tolerance or thermostability than a wildtype pfu polymerase.
2. A dna polymerase of
a first consensus sequence selected from the group consisting of
(1)
XXLXXXXXXXEGXRXXXXXXVXXXXXDXXXTXXXXXXXXXXVVKXXXXXV LIXXXXXNXXXAXXKXXCXXXXXNFALXXXXXXXXXXXXIXXMXXRFXXXX XXXXXXXXXPXXRXXXXXXXXXXXXXXXXVXXQXXXXXXXEXXTTXXXT (SEQ ID NO:30), wherein X is any amino acid or a peptide bond;
(2)
XXEXXXXYXXXXEXXFXXXXKXXXAXXXXXXXXAXXXXTVXTVKRXXXXQ XXXXXRXVEXXXXFTXXXXXXAXXDXIXXXXX (SEQ ID NO:31), wherein X is any amino acid or a peptide bond;
(3)
XXXXXXXXXXXXXXXXALXXDXXXXKXXXXXXXXTEXXSKXXVXXXXXVX HXXXXXDXKDXXXTXXXXXXXXRXXXRXXXXRXXTXXSXXXXKXSXRXGD XXXPFDXFXXTXXXXXXXXXXXXXXXXXXEXXXRAXX (SEQ ID NO:32), wherein X is any amino acid or a peptide bond;
(4)
NGX1FKIEX2DRTFX3PYX4YALLX5DDSX6IEEVKKITX7ERHGX8X9VX10X11X12X13 VEKVX14KKFLGX15PX16X17VWKLYX18X19HPQDVPX20IRX21KX22REHPA (SEQ ID NO:33), wherein X1 is not K; X2 is not H; X3 is not R; X4 is not I; X5 is not R; X6 is not K; X7 is not G; X8 is not K; X9 is not I; X10 is not R; X11 is not I; X12 is not V; X13 is not D; X14 is not E; X15 is not K; X16 is not I; X17 is not T; X18 is not L; X19 is not E; X20 is not T; X21 is not E; and X22 is not V;
(5)
PIX1MISYADEX2X3AX4VITWKNX5DLPYVX6VVSX7EREMIKRFLRX8X9X10EKDPDX11X12X13TYNGDX14FDFX15YLX16KRX17EKLGIX18X19X20X21GRDGSEPKX22QRX23GDX24X25AVEVKGRIHFDLYX26VIX27RTINLPTYTLEAVYEAX28FGX29PKEKVYAX30EIX31X32AWEX33 (SEQ ID NO:34), wherein X1 is not I; X2 is not N; X3 is not E; X4 is not K; X5 is not I; X6 is not E; X7 is not S; X8 is not I; X9 is not I; X10 is not R; X11 is not I; X12 is not I; X13 is not V; X14 is not S; X15 is not P; X16 is not A; X17 is not A; X18 is not K; X19 is not L; X20 is not T; X21 is not I; X22 is not M; X23 is not I; X24 is not M; X25 is not T; X26 is not H; X27 is not T; X28 is not I; X29 is not K; X30 is not D; X31 is not A; X32 is not K; and X33 is not S; and
(6)
RDWSEIAKETQARVLEX1X2LKX3GDVEX4AVRIVKEVX5X6KLX7X8YEX9PPEKLX10 IX11EQITRX12LX13X14YKAX15GPHVAVAKX16LAAX17GVKIX18PGX19VIX20YIVLX21GX22GX23IX24X25RAIX26X27X28EX29DPX30KHKYDAEYYIENQVLPAVX31RILX32X33FG (SEQ ID NO:35), wherein X1 is not T; X2 is not I; X3 is not H; X4 is not E; X5 is not I; X6 is not Q; X7 is not A; X8 is not N; X9 is not I; X10 is not A; X11 is not Y; X12 is not P; X13 is not H; X14 is not E; X15 is not I; X16 is not K; X17 is not K; X18 is not K; X19 is not M; X20 is not G; X21 is not R; X22 is not D; X23 is not P; X24 is not S; X25 is not N; X26 is not L; X27 is not A; X28 is not E; X29 is not Y; X30 is not K; X31 is not L; X32 is not E; and X33 is not G;
and a second consensus sequence selected from the group consisting of
(1)
XKXXXXXXXXXXXXAXXXXXXXXXXXXXXXXXLXXXXNXXIXXXXXXKXX XXIXXXXXXXXXHXXXXXXXXXTXXXEXQXXXXKIXXXXXXKXXXLXXXXF XXXXXXXKXXXXXXXXXXXXXXXXXKXXELVWXXLXXXFXXXXLXIXXXXL YXXXXXGESXEIXXXXLX (SEQ ID NO:36), wherein X is any amino acid or a peptide bond;
(2)
EX1GLWENIVYLDFRX2LYPSIIITHNVSPDTLNX3EGCKX4YDX5APQVGHX6FCKDX7PGFIPSLLGX8LLEERQKIKX0KMKX10TX11DPIEX12X13LLDYRQX14AIKX15LANSX16YGYYGYAX17ARWYCKECAESVTAWGRX18YIX19X20X21X22KEX23EEKX24GFKVX25YX26DTDGX27X28ATIPGX29X30X31EX32X33KKKAX34E (SEQ ID NO:37), wherein X1 is not R; X2 is not S; X3 is not R; X4 is not E; X5 is not V; X6 is not R; X7 is not F; X8 is not D; X9 is not K; X10 is not A; X11 is not I; X12 is not R; X13 is not K; X14 is not R; X15 is not I; X16 is not Y; X17 is not R; X18 is not E; X19 is not T; X20 is not M; X21 is not T; X22 is not I; X23 is not I; X24 is not Y; X25 is not I; X26 is not S; X27 is not F; X28 is not F; X29 is not A; X30 is not D; X31 is not A; X32 is not T; X33 is not V; X34 is not M.
4. The dna polymerase of
XXXXTXXXXXDXXXXXXIXXXXXXEXXXXYXXXXEXXFXXXXFXXXAXXXXXXXX AXXXXTVXTVKRXXXXQXXXXXRXVEXXXXXFTXXXXXXAXXDXIXXXXXXIXXYX XXXXXXXXXXXXXXXVXXXXDXXXXMXXXXXXXXXXXXXXXAEXXXLXXXXXXX EGXRXXXXXXVXXXXXDXXXTXXXXXXXXXXVVKXXXXVLIXXXXXNXXXAXXK XXCXXXXXNFALXXXXXXXXXXIXXMXXRFXXXXXXXXXXXXXPXXRXXXXXXXX XXXXXXXXVXXQXXXXXXXEXXTTXXXTXXXXXXXXRXXXXXXXVXXXXXXXXX XXXAXXXXXVXXPXXXXXXXXXXXXXXXXXXXXXXXXXXVXXXXXSXEXYQXXX XEXXTXXFXXXXXKXXXXXXXXXXXXAXXXXXXXXXXXXXXXXXLXXXXNXXIXX XXXXKXXXXIXXXXXXXXXHXXXXXXXXXTXXXEXQXXXXKIXXXXXXKXXXLXX XXFXXXXXXXKXXXXXXXXXXXXXXXXXKXXELVWXXLXXXFXXXXLXIXXXXLY XXXXXGESXEIXXXXLXXLXXXXAXXXXAXXXXXXXXXXXXXXXXXXKXXXXXXX XXITXXXXXXXXXXXXXXXXXXXXXXXALXXDXXXXKXXXXXXXXTEXXSKXXV XXXXXVXHXXXXXDXKDXXXTXXXXXXXXRXXXRXXXXRXXTXXSXXXXKXSXRX GDXXXPFDXFXXTXXXXXXXXXXXXXXXXXXEXXXRAXXXXXXXXXXXXXXXXXX SAXXKPXGT (SEQ ID NO:38)
wherein X is any amino acid or a peptide bond.
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The present application is a national phase entry of international application Ser. No. PCT/US2009/063166, filed Nov. 3, 2009 which claims priority to U.S. Provisional patent application Ser. No. 61/110,862, filed Nov. 3, 2008, the entire disclosure of which is incorporated herein by reference.
The present specification makes reference to a Sequence Listing (submitted electronically as a .txt file named “Sequence Listing.txt” on May 3, 2011). The .txt file was generated on Nov. 9, 2009 and is 235 kb in size. The entire contents of the Sequence Listing are herein incorporated by reference.
DNA polymerases are enzymes that use single-stranded DNA as a template to synthesize the complementary DNA strand. In particular, DNA polymerases can add free nucleotides to the 3′ end of a newly-forming strand resulting in elongation of the new strand in a 5′-3′ direction. Some DNA polymerases can correct mistakes in newly-synthesized DNA. This process is known as error correction. These polymerases can recognize an incorrectly incorporated nucleotide and the 3′->5′ exonuclease activity of the enzyme allows the incorrect nucleotide to be excised (this activity is known as proofreading). Following base excision, the polymerase can re-insert the correct base and replication can continue. The proofreading function gives the DNA replication much higher fidelity than it would have if synthesis were the result of only a base-pairing selection step. Brutlag, D. and Kornberg, A., J. Biol. Chem., 247:241-248 (1972). DNA polymerases with 3′-5′ proofreading exonuclease activity have a substantially lower error rate when compared with a non-proofreading exonuclease-possessing polymerase. Chang, L. M. S., J. Biol. Chem., 252:1873-1880 (1977). However, sometimes, the advantage of these polymerases is offset by its relatively low processivity that reduces the yield of DNA amplification products.
The present invention encompasses the discovery that domain swapping can combine desired functional characteristics (e.g., high processivity, high elongation rate, thermostability, resistance to salt, PCR additives (e.g., PCR enhancers) and other impurities, and high fidelity) of different DNA polymerases in a chimeric enzyme. Thus, the present invention provides, among other things, robust, fast and accurate DNA polymerases for DNA amplification, synthesis, detection, sequencing and other important recombinant DNA techniques.
In one aspect, the present invention provides chimeric polymerases containing a first domain having a sequence at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to an amino acid sequence found in a first DNA polymerase characterized with high processivity, elongation rate, salt resistance, thermostability or TMAC tolerance; and a second domain having a sequence at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to an amino acid sequence found in a second DNA polymerase characterized with high fidelity, wherein the chimeric polymerases are characterized with both high fidelity and high processivity, elongation rate, or salt resistance. As used herein, the term “high processivity” refers to a processivity higher than 20 nts (e.g., higher than 40 nts, 60 nts, 80 nts, 100 nts, 120 nts, 140 nts, 160 nts, 180 nts, 200 nts, 220 nts, 240 nts, 260 nts,280 nts, 300 nts, 320 nts, 340 nts, 360 nts, 380 nts, 400 nts, or higher) per association/disassociation with the template. As used herein, the term “high elongation rate” refers to an elongation rate higher than 25 nt/s (e.g., higher than 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140 nt/s). As used herein, the term “high salt resistance” refers to the ability of a DNA polymerase to substantially maintain its enzymatic activity at a salt concentration higher than 30 mM (e.g., higher than 35 mM, 40 mM, 45 mM, or 50 mM). As used herein, the term “high fidelity” refers to an error rate less than 4.45×10−6 (e.g., less than 4.0×10−6, 3.5 ×10−6, 3.0×10−6, 2.5×10−6, 2.0×10−6, 1.5×10−6, 1.0×10−6, 0.5×10−6) mutations/nt/doubling. As used herein, the term “high TMAC tolerance” refers to the ability of a DNA polymerase to substantially maintain its enzymatic activity at a TMAC (tetra-methyl ammonium chloride) concentration higher than 10 mM (e.g., higher than 15 mM, 20 mM, 25 mM, 30 mM). As used herein, the term “high thermostability” refers to the ability of a DNA polymerase to substantially maintain its enzymatic activity after more than 30 minutes incubation at 98° C. (e.g., 45 min, 60 min, 90 min, 180 min, 210 min, 240 min). The terms of “processivity,” “elongation rate,” “fidelity,” “salt resistance,” “TMAC tolerance,” and “thermostability” are further defined in the Definitions section.
In some embodiments, exemplary first DNA polymerases suitable for the present invention include, but are not limited to, KOD polymerase, TNA1 polymerase, Thermococcus sp. 9 degrees N-7, T4, T7, or phi29. In some embodiments, the first DNA polymerase is KOD polymerase. In some embodiments, exemplary second DNA polymerases suitable for the invention include, but are not limited to, polymerases isolated from Pyrococcus furiosus, P. abyssi, T. gorgonarius, T. litoralis, T. zilligii, T. sp. GT, or P. sp. GB-D. In some embodiments, the second DNA polymerase is Pfu polymerase. In particular embodiments, the first DNA polymerase is KOD polymerase and the second DNA polymerase is Pfu polymerase.
In some embodiments, suitable first domain is an exonuclease domain, N-terminal domain, and/or a thumb domain. In some embodiments, suitable second domain is palm and/or fingers domain.
In some embodiments, amino acid sequences found in the first DNA polymerase correspond to amino acid residues 26 to 105 of KOD polymerase (SEQ ID NO:11), amino acid residues 156 to 301 of KOD polymerase (SEQ ID NO:11), and/or amino acid residues 612 to 749 of KOD polymerase (SEQ ID NO:11).
In some embodiments, amino acid sequences found in the second DNA polymerase correspond to amino acid residues 394 to 563 of Pfu polymerase (SEQ ID NO:9).
In some embodiments, a chimeric polymerase in accordance with the present invention include a first domain having a consensus sequence selected from the group consisting of
In some embodiments, chimeric polymerases in accordance with the present invention are defined by consensus sequence XXXXTXXXXXDXXXXXXIXXXXXXEXXXXYXXXXEXXFXXXXKXXXAXXXXXX XXAXXXXTVXTVKRXXXXQXXXXXRXVEXXXXXFTXXXXXXAXXDXIXXXXXXI XXYXXXXXXXXXXXXXXXXVXXXXDXXXXMXXXXXXXXXXXXXXXAEXXXLX XXXXXXEGXRXXXXXXVXXXXXDXXXTXXXXXXXXXXVVKXXXXXVLIXXXXX NXXXAXXKXXCXXXXXNFALXXXXXXXXXXIXXMXXRFXXXXXXXXXXXXPX XRXXXXXXXXXXXXXXXXVXXQXXXXXXXEXXTTXXXTXXXXXXXXRXXXXX XXVXXXXXXXXXXXXAXXXXXVXXPXXXXXXXXXXXXXXXXXXXXXXXXXXV XXXXXSXEXYQXXXXEXXTXXFXXXXXKXXXXXXXXXXXXAXXXXXXXXXXXX XXXXXLXXXXNXXIXXXXXXKXXXXIXXXXXXXXXHXXXXXXXXXTXXXEXQX XXXKIXXXXXXKXXXLXXXXFXXXXXXXKXXXXXXXXXXXXXXXXXKXXELVW XXLXXXFXXXXLXIXXXXLYXXXXXGESXEIXXXXLXXLXXXXAXXXXAXXXXX XXXXXXXXXXXXXKXXXXXXXXXITXXXXXXXXXXXXXXXXXXXXXXXXALX XKXXXXKXXXXXXXXTEXXSKXXVXXXXXVXHXXXXXDXKDXXXTXXXXXXX XRXXXRXXXXRXXTXXTXXSXXXXKXSXRXGDXXXPFDXFXXTXXXXXXXXXXXXX XXXXXEXXXRAXXXXXXXXXXXXXXXXXXSAXXKPXGT (SEQ ID NO:38), wherein X is any amino acid or a peptide bond, and wherein the chimeric polymerase has a fidelity higher than that of KOD and a processivity, an elongation rate, a salt resistance, a TMAC or other PCR enhancer tolerance or a thermostability higher than that of Pfu.
In some embodiments, chimeric polymerases in accordance with the present invention are defined by consensus sequence XIXDTDYXTXDGXPXXRIFXKXXGEFXXXYDXXEPYFYALLKDDSAIXXXXXXXA XRHGTVXTVKRXXXXQXKFLXRXVEVWXLXFTHPQDVPAXXDXIXXHXXVIDIYE YDIPFAKRYLIDXGLVPMEGDEXLXMXXXDIETXYHEGXEFAEGXXLMISYADXEG ARVITWKXVDLPYVDVVSTEXEMIKRXXXVVKEKDPDVLIXYXGDNEDXAYLKXR CEXLGXNFALXRXXXXXEPKIXXMGXRFAVEXXGRXHFDLXPXXRXTXNLPTYXL XXVYEXVSGQXKXKXXXXEEITTXWETXXXXXXXARYSMEDAXYTXELGXEFXPM EAXLXXLVGXPXWDVXRSSTGNLVEWXLLXXAYXRNEVAPNKPSXEEYQXRXXE XYTGXFVXEPEKGLWXXXXXLDXXALYPSIIIXXHNVSPDTLXLEXCXNYDIAPXVG XKFCKDIPGFIPSXLXHLXXXRQXXKTXMXEXQDPXEKIXLDYRQKAXKLLXNSFY GYXGYXKARWYXXECAESVTXWGRKYIELVWXELEXXFGFKXLYIDTDGLYATIP GGESXEIKXXXLXFLXYINAXLPGALELEYEXFYXRGFFVXKKKYAXIDEEXXITTR GLEXVRRDWSXXAKETXAXVLEALLXDXXVXKAVXXVXXXTEXXSKYXVPXEKL VIHEQITRDXKDYXATGPHVAXAKRLXXGXXXRPGTXISYXXLKGSGRXGDRXIPF DEFXXTKHXYDXXYYIENQVLPAVERXLRAFGYXXXXLXXQXXXQXGLSAWXKP XGT (SEQ ID NO:39), wherein X is any amino acid or a peptide bond.
In some embodiments, the present invention further provides chimeric polymerases containing a first domain having a sequence at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to an amino acid sequence found in an exonuclease domain, an N-terminal domain, and/or a thumb domain of a first DNA polymerase; and a second domain having a sequence at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to an amino acid sequence found in palm and/or fingers domain of a second DNA polymerase. In some embodiments, the chimeric polymerase has a fidelity higher than that of the second DNA polymerase and a processivity, an elongation rate, a salt resistance, a TMAC or other PCR enhancer tolerance or a thermostability higher than that of the first DNA polymerase.
In another aspect, the present invention provides methods of engineering chimeric polymerases. Inventive methods in accordance with the present invention include steps of: (a) providing an N-terminal domain, an exonuclease domain, and/or a thumb domain based on a first DNA polymerase; (b) providing a palm and/or fingers domain based on a second DNA polymerase; (c) combining the domains from step (a) and step (b) to form a chimeric polymerase; wherein the chimeric polymerase has a fidelity higher than that of the first DNA polymerase and a processivity, an elongation rate, a salt resistance, a TMAC or other PCR enhancer tolerance or a thermostability higher than that of the second DNA polymerase. In some embodiments, a chimeric polymerase engineered according to the present invention has a processivity, an elongation rate, a salt resistance, a TMAC or other PCR enhancer tolerance or a thermostability substantially similar to that of the first DNA polymerase and a fidelity substantially similar to that of the second DNA polymerase.
In some embodiments, exemplary first DNA polymerases suitable for the present invention include, but are not limited to, KOD polymerase, TNA1 polymerase, Thermococcus sp. 9 degrees N-7, T4, T7, or phi29. In some embodiments, the first DNA polymerase is KOD polymerase. In some embodiments, exemplary second DNA polymerases suitable for the invention include, but are not limited to, polymerases isolated from Pyrococcus furiosus, P. abyssi, T. gorgonarius, T. litoralis, T. zilligii, T. sp. GT, or P. sp. GB-D. In some embodiments, the second DNA polymerase is Pfu polymerase.
In some embodiments, the first DNA polymerase is KOD polymerase and the second DNA polymerase is Pfu polymerase. In some embodiments, the first DNA polymerase is Pfu polymerase and the second DNA polymerase is KOD polymerase.
In some embodiments, the present invention provides methods of improving the fidelity of a DNA polymerase. In particular embodiments, inventive methods in accordance with the invention include a step of replacing a sequence within the palm and/or fingers domain of the DNA polymerase of interest with a corresponding sequence from a different DNA polymerase that is characterized with higher fidelity relative to the DNA polymerase of interest.
In some embodiments, the present invention provides methods of improving the processivity, elongation rate, salt resistance, TMAC or other PCR enhancer tolerance or thermostability of a DNA polymerase. In particular embodiments, inventive methods in accordance with the present invention include a step of replacing a sequence within the N-terminal domain, the exonuclease domain and/or the thumb domain of the DNA polymerase of interest with a corresponding sequence from a different DNA polymerase that is characterized with higher processivity, elongation rate, salt resistance, TMAC or other PCR enhancer tolerance or thermostability relative to the DNA polymerase of interest.
The present invention provides various chimeric polymerases described herein including chimeric polymerases engineered and/or improved using inventive methods as described herein. In some embodiments, chimeric polymerases in accordance with the present invention contain an amino acid sequence at least 80% identical to SEQ ID NO:16 (the Kofu amino acid sequence as shown in the Sequences section). In particular embodiments, a chimeric polymerase in accordance with the present invention contains the amino acid sequence of SEQ ID NO:16. In some embodiments, chimeric polymerases in accordance with the present invention contain an amino acid sequence at least 80% identical to SEQ ID NO:15 (the Pod amino acid sequence as shown in the Sequences section). In particular embodiments, a chimeric polymerase in accordance with the present invention contains the amino acid sequence of SEQ ID NO:15.
The present invention also provides kits and compositions containing various chimeric polymerases described herein and uses thereof (e.g., methods of amplifying DNA fragments using chimeric DNA polymerases of the invention). In addition, the present invention provides nucleotide sequences encoding various chimeric polymerases described herein and vectors and/or cells containing the nucleotide sequences according to the invention.
The drawings are for illustration purposes only, not for limitation.
Amino acid: As used herein, term “amino acid,” in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2N—C(H)(R)—COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. As used herein, “synthetic amino acid” encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions. Amino acids, including carboxy- and/or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, and/or substitution with other chemical groups. Amino acids may participate in a disulfide bond. The term “amino acid” is used interchangeably with “amino acid residue,” and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide. It should be noted that all amino acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus.
Base Pair (bp): As used herein, base pair refers to a partnership of adenine (A) with thymine (T), or of cytosine (C) with guanine (G) in a double stranded DNA molecule.
Chimeric polymerase: As used herein, the term “chimeric polymerase” (also referred to as “chimera”) refers to any polymerase containing two or more heterologous domains, amino acid sequences, peptides, and/or proteins joined either covalently or non-covalently to produce a polymerase that does not occur in nature. Typically, a chimeric polymerase contains a first domain joined to a second domain, wherein the first and second domains are not found in the same relationship in nature. Typically, the first domain is derived from a first DNA polymerase and a second domain is derived from a second DNA polymerase. Typically, the first and second DNA polymerases are characterized with at least one distinct functional characteristics (e.g., processivity, elongation rate, fidelity, salt tolerance, tolerance to PCR additives or thermostability). As used herein, a sequence derived from a DNA polymerase of interest refers to any sequence found in the DNA polymerase of interest, or any sequence having at least 70% (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to an amino acid sequence found in the DNA polymerase of interest. A “chimeric polymerase” according to the invention may contain two or more amino acid sequences from related or similar polymerases (e.g., proteins sharing similar sequences and/or structures), joined to form a new functional protein. A “chimeric polymerase” according to the invention may contain two or more amino acid sequences from unrelated polymerases, joined to form a new functional protein. For example, a chimeric polymerase of the invention may be an “interspecies” or “intergenic” fusion of protein structures expressed by different kinds of organisms.
Complementary: As used herein, the term “complementary” refers to the broad concept of sequence complementarity between regions of two polynucleotide strands or between two nucleotides through base-pairing. It is known that an adenine nucleotide is capable of forming specific hydrogen bonds (“base pairing”) with a nucleotide which is thymine or uracil. Similarly, it is known that a cytosine nucleotide is capable of base pairing with a guanine nucleotide.
DNA binding affinity: As used herein, the term “DNA-binding affinity” typically refers to the activity of a DNA polymerase in binding DNA nucleic acid. In some embodiments, DNA binding activity can be measured in a two band-shift assay. For example, in some embodiments (based on the assay of Guagliardi et al. (1997) J. Mol. Biol. 267:841-848), double-stranded nucleic acid (the 452-bp HindIII-EcoRV fragment from the S. solfataricus lacS gene) is labeled with 32P to a specific activity of at least about 2.5×107 cpm/μg (or at least about 4000 cpm/fmol) using standard methods. See, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, NY) at 9.63-9.75 (describing end-labeling of nucleic acids). A reaction mixture is prepared containing at least about 0.5 μg of the polypeptide in about 10 μl of binding buffer (50 mM sodium phosphate buffer (pH 8.0), 10% glycerol, 25 mM KCl, 25 mM MgCl2). The reaction mixture is heated to 37° C. for 10 min. About 1×104 to 5×104 cpm (or about 0.5-2 ng) of the labeled double-stranded nucleic acid is added to the reaction mixture and incubated for an additional 10 min. The reaction mixture is loaded onto a native polyacrylamide gel in 0.5× Tris-borate buffer. The reaction mixture is subjected to electrophoresis at room temperature. The gel is dried and subjected to autoradiography using standard methods. Any detectable decrease in the mobility of the labeled double-stranded nucleic acid indicates formation of a binding complex between the polypeptide and the double-stranded nucleic acid. Such nucleic acid binding activity may be quantified using standard densitometric methods to measure the amount of radioactivity in the binding complex relative to the total amount of radioactivity in the initial reaction mixture. Other methods of measuring DNA binding affinity are known in the art (see, e.g., Kong et al. (1993) J. Biol. Chem. 268(3):1965-1975).
Domain: As used herein, the term “Domain” as used herein refers to an amino acid sequence of a polypeptide (e.g., polymerase) comprising one or more defined functions or properties.
Elongation rate: As used herein, the term “elongation rate” refers to the average speed at which a DNA polymerase extends a polymer chain. As used herein, a high elongation rate refers to an elongation rate higher than 25 nt/s (e.g., higher than 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140 nt/s).
Enzyme activity: As used herein, the term “enzyme activity” refers to the specificity and efficiency of a DNA polymerase. Enzyme activity of a DNA polymerase is also referred to as “polymerase activity,” which typically refers to the activity of a DNA polymerase in catalyzing the template-directed synthesis of a polynucleotide. Enzyme activity of a polymerase can be measured using various techniques and methods known in the art. For example, serial dilutions of polymerase can be prepared in dilution buffer (e.g., 20 mM Tris.Cl, pH 8.0, 50 mM KCl, 0.5% NP 40, and 0.5% Tween-20). For each dilution, 5 μl can be removed and added to 45 μl of a reaction mixture containing 25 mM TAPS (pH 9.25), 50 mM KCl, 2 mM MgCl2, 0.2 mM dATP, 0.2 mM dGTP, 0.2 mM dTTP, 0.1 mM dCTP, 12.5 μg activated DNA, 100 μM [α-32P]dCTP (0.05 μCi/nmol) and sterile deionized water. The reaction mixtures can be incubated at 37° C. (or 74° C. for thermostable DNA polymerases) for 10 minutes and then stopped by immediately cooling the reaction to 4° C. and adding 10 μl of ice-cold 60 mM EDTA. A 25 μl aliquot can be removed from each reaction mixture. Unincorporated radioactively labeled dCTP can be removed from each aliquot by gel filtration (Centri-Sep, Princeton Separations, Adelphia, N.J.). The column eluate can be mixed with scintillation fluid (1 ml). Radioactivity in the column eluate is quantified with a scintillation counter to determine the amount of product synthesized by the polymerase. One unit of polymerase activity can be defined as the amount of polymerase necessary to synthesize 10 nmole of product in 30 minutes (Lawyer et al. (1989) J. Biol. Chem. 264:6427-647). Other methods of measuring polymerase activity are known in the art (see, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, NY)).
Fidelity: As used herein, the term “fidelity” refers to the accuracy of DNA polymerization by template-dependent DNA polymerase. The fidelity of a DNA polymerase is typically measured by the error rate (the frequency of incorporating an inaccurate nucleotide, i.e., a nucleotide that is not complementary to the template nucleotide). The accuracy or fidelity of DNA polymerization is maintained by both the polymerase activity and the 3′-5′ exonuclease activity of a DNA polymerase. The term “high fidelity” refers to an error rate less than 4.45×10−6 (e.g., less than 4.0×10−6, 3.5×10−6, 3.0×10−6, 2.5×10−6, 2.0×10−6, 1.5×10−6, 1.0×10−6, 0.5×10−6) mutations/nt/doubling. The fidelity or error rate of a DNA polymerase may be measured using assays known to the art. For example, the error rates of DNA polymerases can be tested using the lacI PCR fidelity assay described in Cline, J. et al. (1996) NAR 24: 3546-3551. Briefly, a 1.9 kb fragment encoding the lacIOlacZa target gene is amplified from pPRIAZ plasmid DNA using 2.5 U DNA polymerase (i.e., amount of enzyme necessary to incorporate 25 nmoles of total dNTPs in 30 min. at 72° C.) in the appropriate PCR buffer. The lad-containing PCR products are then cloned into lambda GT10 arms, and the percentage of lacI mutants (MF, mutation frequency) is determined in a color screening assay, as described (Lundberg, K. S., Shoemaker, D. D., Adams, M. W. W., Short, J. M., Sorge, J. A., and Mathur, E. J. (1991) Gene 180: 1-8). Error rates are expressed as mutation frequency per by per duplication (MF/bp/d), where by is the number of detectable sites in the lad gene sequence (349) and d is the number of effective target doublings. Similar to the above, any plasmid containing the lacIOlacZa target gene can be used as template for the PCR. The PCR product may be cloned into a vector different from lambda GT (e.g., plasmid) that allows for blue/white color screening.
Joined: As used herein, “joined” refers to any method known in the art for functionally connecting polypeptide domains, including without limitation recombinant fusion with or without intervening domains, inter-mediated fusion, non-covalent association, and covalent bonding, including disulfide bonding, hydrogen bonding, electrostatic bonding, and conformational bonding.
Nucleotide: As used herein, a monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1′ carbon of the pentose) and that combination of base and sugar is a nucleoside. When the nucleoside contains a phosphate group bonded to the 3′ or 5′ position of the pentose it is referred to as a nucleotide. A sequence of operatively linked nucleotides is typically referred to herein as a “base sequence” or “nucleotide sequence,” and is represented herein by a formula whose left to right orientation is in the conventional direction of 5′-terminus to 3′-terminus.
Oligonucleotide or Polynucleotide: As used herein, the term “oligonucleotide” is defined as a molecule including two or more deoxyribonucleotides and/or ribonucleotides, preferably more than three. Its exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotide may be derived synthetically or by cloning. As used herein, the term “polynucleotide” refers to a polymer molecule composed of nucleotide monomers covalently bonded in a chain. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are examples of polynucleotides.
Polymerase: As used herein, a “polymerase” refers to an enzyme that catalyzes the polymerization of nucleotide (i.e., the polymerase activity). Generally, the enzyme will initiate synthesis at the 3′-end of the primer annealed to a polynucleotide template sequence, and will proceed towards the 5′ end of the template strand. A “DNA polymerase” catalyzes the polymerization of deoxynucleotides.
Processivity: As used herein, “processivity” refers to the ability of a polymerase to remain attached to the template and perform multiple modification reactions. “Modification reactions” include but are not limited to polymerization, and exonucleolytic cleavage. In some embodiments, “processivity” refers to the ability of a DNA polymerase to perform a sequence of polymerization steps without intervening dissociation of the enzyme from the growing DNA chains. Typically, “processivity” of a DNA polymerase is measured by the length of nucleotides (for example 20 nts, 300 nts, 0.5-1 kb, or more) that are polymerized or modified without intervening dissociation of the DNA polymerase from the growing DNA chain. “Processivity” can depend on the nature of the polymerase, the sequence of a DNA template, and reaction conditions, for example, salt concentration, temperature or the presence of specific proteins. As used herein, the term “high processivity” refers to a processivity higher than 20 nts (e.g., higher than 40 nts, 60 nts, 80 nts, 100 nts, 120 nts, 140 nts, 160 nts, 180 nts, 200 nts, 220 nts, 240 nts, 260 nts,280 nts, 300 nts, 320 nts, 340 nts, 360 nts, 380 nts, 400 nts, or higher) per association/disassociation with the template. Processivity can be measured according the methods defined herein and in WO 01/92501 A1.
Primer: As used herein, the term “primer” refers to an oligonucleotide, whether occurring naturally or produced synthetically, which is capable of acting as a point of initiation of nucleic acid synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, e.g., in the presence of four different nucleotide triphosphates and thermostable enzyme in an appropriate buffer (“buffer” includes appropriate pH, ionic strength, cofactors, etc.) and at a suitable temperature. The primer is preferably single-stranded for maximum efficiency in amplification, but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the thermostable enzyme. The exact lengths of the primers will depend on many factors, including temperature, source of primer and use of the method. For example, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 nucleotides, although it may contain more or few nucleotides. Short primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with template.
Salt resistance: As used herein, the term “salt resistance” (also referred to as salt tolerance) refers to the ability of a DNA polymerase to substantially maintain its enzymatic activity in the presence of salt or PCR additives (e.g., TMAC). In some embodiments, resistance to salt or PCR additives is measured by the maximum salt concentration at which a DNA polymerase is still active. The maximum salt concentration differs for each polymerase and is known in the art, or can be experimentally determined according to methods in the art. For example, Pfu is inhibited at 30 mM salt (in a PCR reaction).
Synthesis: As used herein, the term “synthesis” refers to any in vitro method for making new strand of polynucleotide or elongating existing polynucleotide (i.e., DNA or RNA) in a template dependent manner. Synthesis, according to the invention, includes amplification, which increases the number of copies of a polynucleotide template sequence with the use of a polymerase. Polynucleotide synthesis (e.g., amplification) results in the incorporation of nucleotides into a polynucleotide (i.e., a primer), thereby forming a new polynucleotide molecule complementary to the polynucleotide template. The formed polynucleotide molecule and its template can be used as templates to synthesize additional polynucleotide molecules. “DNA synthesis,” as used herein, includes, but is not limited to, PCR, the labeling of polynucleotide (i.e., for probes and oligonucleotide primers), polynucleotide sequencing.
Template DNA molecule: As used herein, the term “template DNA molecule” refers to a strand of a nucleic acid from which a complementary nucleic acid strand is synthesized by a DNA polymerase, for example, in a primer extension reaction.
Template dependent manner: As used herein, the term “template dependent manner” refers to a process that involves the template dependent extension of a primer molecule (e.g., DNA synthesis by DNA polymerase). The term “template dependent manner” typically refers to polynucleotide synthesis of RNA or DNA wherein the sequence of the newly synthesized strand of polynucleotide is dictated by the well-known rules of complementary base pairing (see, for example, Watson, J. D. et al., In: Molecular Biology of the Gene, 4th Ed. , W. A. Benjamin, Inc., Menlo Park, Calif. (1987)).
Thermostable enzyme: As used herein, the term “thermostable enzyme” refers to an enzyme which is stable to heat (also referred to as heat-resistant) and catalyzes (facilitates) polymerization of nucleotides to form primer extension products that are complementary to a polynucleotide template sequence. Typically, thermostable stable polymerases are preferred in a thermocycling process wherein double stranded nucleic acids are denatured by exposure to a high temperature (e.g., about 95 C) during the PCR cycle. A thermostable enzyme described herein effective for a PCR amplification reaction satisfies at least one criteria, i.e., the enzyme do not become irreversibly denatured (inactivated) when subjected to the elevated temperatures for the time necessary to effect denaturation of double-stranded nucleic acids. Irreversible denaturation for purposes herein refers to permanent and complete loss of enzymatic activity. The heating conditions necessary for denaturation will depend, e.g., on the buffer salt concentration and the length and nucleotide composition of the nucleic acids being denatured, but typically range from about 90° C. to about 98° C. for a time depending mainly on the temperature and the nucleic acid length, typically about 0.2 to four minutes. Higher temperatures may be tolerated as the buffer salt concentration and/or GC composition of the nucleic acid is increased. In some embodiments, thermostable enzymes will not become irreversibly denatured at about 90° C. -100° C. Typically, a thermostable enzyme suitable for the invention has an optimum temperature at which it functions that is higher than about 40° C., which is the temperature below which hybridization of primer to template is promoted, although, depending on (1) magnesium and salt, concentrations and (2) composition and length of primer, hybridization can occur at higher temperature (e.g., 45° C.-70° C.). The higher the temperature optimum for the enzyme, the greater the specificity and/or selectivity of the primer-directed extension process. However, enzymes that are active below 40° C. (e.g., at 37° C.) are also with the scope of this invention provided they are heat-stable. In some embodiments, the optimum temperature ranges from about 50° C. to 90° C. (e.g., 60° C.-80° C.).
TMAC or other PCR enhancer tolerance: As used herein, the term “TMAC or other PCR enhancer tolerance” (also referred to as TMAC or other PCR enhancer resistance) refers to the ability of a DNA polymerase to substantially maintain its enzymatic activity in the presence of TMAC or other PCR enhancers (e.g., glycerol, DMSO, betaine, amides, other tetramethyl ammonium salts).
The present invention provides, among other things, chimeric DNA polymerases containing heterologous domains having sequences derived from at least two DNA polymerases that have at least one distinct functional characteristics (e.g., elongation rate, processivity, error rate or fidelity, salt tolerance or resistance) and methods of making and using the same.
DNA Polymerases
Chimeric DNA polymerases in accordance with the present invention may be engineered from any DNA polymerases, in particular, thermostable polymerases. Typically, DNA polymerases are grouped into six families: A, B, C, D, X and Y. Families A, B, C are grouped based on their amino acid sequence homologies to E. coli polymerases I, II, and III, respectively. Family X has no homologous E. coli polymerases. In some embodiments, DNA polymerases suitable for the present invention are family B DNA polymerases. Family B polymerases include, but are not limited to, E. coli pol II, archaeal polymerases, PRD1, phi29, M2, T4 bacteriophage DNA polymerases, eukaryotic polymerases α, Δ, ε, and many viral polymerases. In some embodiments, DNA polymerases suitable for the invention are archaeal polymerases (e.g., euryarchaeal polymerases).
Suitable exemplary archaeal polymerases include, but are not limited to, DNA polymerases from archaea (e.g., Thermococcus litoralis (Vent™, GenBank: AAA72101), Pyrococcus furiosus (Pfu, GenBank: D12983, BAA02362), Pyrococcus woesii, Pyrococcus GB-D (Deep Vent™, GenBank: AAA67131), Thermococcus kodakaraensis KODI (KOD, GenBank: BD175553, BAA06142; Thermococcus sp. strain KOD (Pfx, GenBank: AAE68738)), Thermococcus gorgonarius (Tgo, Pdb: 4699806), Sulfolobus solataricus (GenBank: NC002754, P26811), Aeropyrum pernix (GenBank: BAA81109), Archaeglobus fulgidus (GenBank: 029753), Pyrobaculum aerophilum (GenBank: AAL63952), Pyrodictium occultum (GenBank: BAA07579, BAA07580), Thermococcus 9 degree Nm (GenBank: AAA88769, Q56366), Thermococcus fumicolans (GenBank: CAA93738, P74918), Thermococcus hydrothermalis (GenBank: CAC18555), Thermococcus sp. GE8 (GenBank: CAC12850), Thermococcus sp. JDF-3 (GenBank: AX135456; WO0132887), Thermococcus sp. TY (GenBank: CAA73475), Pyrococcus abyssi (GenBank: P77916), Pyrococcus glycovorans (GenBank: CAC12849), Pyrococcus horikoshii (GenBank: NP 143776), Pyrococcus sp. GE23 (GenBank: CAA90887), Pyrococcus sp. ST700 (GenBank: CAC 12847), Thermococcus pacificus (GenBank: AX411312.1), Thermococcus zilligii (GenBank: DQ3366890), Thermococcus aggregans, Thermococcus barossii, Thermococcus celer (GenBank: DD259850.1), Thermococcus profundus (GenBank: E14137), Thermococcus siculi (GenBank: DD259857.1), Thermococcus thioreducens, Thermococcus onnurineus NA1, Sulfolobus acidocaldarium, Sulfolobus tokodaii, Pyrobaculum calidifontis, Pyrobaculum islandicum (GenBank: AAF27815), Methanococcus jannaschii (GenBank: Q58295), Desulforococcus species TOK, Desulfurococcus, Pyrolobus, Pyrodictium, Staphylothermus, Vulcanisaetta, Methanococcus (GenBank: P52025) and other archaeal B polymerases, such as GenBank AAC62712, P956901, BAAA07579)). Additional representative temperature-stable family A and B polymerases include, e.g., polymerases extracted from the thermophilic bacteria Thermus species (e.g., flavus, ruber, thermophilus, lacteus, rubens, aquaticus), Bacillus stearothermophilus, Thermotoga maritima, Methanothermus fervidus.
DNA polymerases suitable for the present invention include DNA polymerases that have not yet been isolated. Suitable polymerases for the present invention include fusion polymerases. Fusion polymerases generally contain an additional protein domain at the N- or C-terminus that changes the phenotype of the fusion polymerase compared to the polymerase without the extra domain. Exemplary polymerases include, but are not limited to, polymerases with double-stranded DNA-binding domains fused at the C- or N-terminus. Further examples of fusion polymerases include those with dUTPase fused to the N-or C-terminus (U.S. patent application 20070190538).
In some embodiments, chimeric DNA polymerases according to the invention contain sequences derived from two or more DNA polymerases that have at least one distinct functional characteristic. Exemplary functional characteristics include, but are not limited to, processivity, elongation rate, fidelity, resistance to salt or PCR additive (e.g., PCR enhancers), thermostability, strand displacement activity, exonuclease activity, uracil read-ahead function, nucleotide selectivity, ability to incorporate modified analogs, and reverse transcriptase activity. For example, some DNA polymerases are characterized with high fidelity. As used herein, the term “high fidelity” refers to an error rate less than 4.45×10−6 (e.g., less than 4.0×10−6, 3.5×10−6, 3.0×10−6, 2.5×10−6, 2.0×10−6, 1.5×10−6, 1.0×10−6, 0.5×10−6) mutations/nt/doubling. Some DNA polymerases are characterized with high processivity. As used herein, the term “high processivity” refers to a processivity higher than 20 nts (e.g., higher than 40 nts, 60 nts, 80 nts, 100 nts, 120 nts, 140 nts, 160 nts, 180 nts, 200 nts, 220 nts, 240 nts, 260 nts,280 nts, 300 nts, 320 nts, 340 nts, 360 nts, 380 nts, 400 nts, or higher) per association/disassociation with the template. Some DNA polymerases are characterized with high elongation rate. As used herein, the term “high elongation rate” refers to an elongation rate higher than 25 nt/s (e.g., higher than 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140 nt/s). Some enzymes are characterized with high resistance to salt (also referred to as salt tolerance). As used herein, the term “high resistance to salt” (also referred to as high salt tolerance) refers to the ability of a DNA polymerase to substantially maintain its activity at a salt concentration higher than 30 mM (e.g., higher than 35 mM, 40 mM, 45 mM, 50 mM). In addition, some enzymes are characterized with resistance to PCR additives. Certain PCR additives are PCR enhancers. For example, Kovarova et al. showed that TMA salts, DMSO, betaine and formamide act as PCR enhancers (Kovarova and Draber. (2000) Nucl. Acids. Res. 28(13), e70). Another example of PCR enhancers is glycerol. Some enzymes are characterized with resistance to PCR enhancers, in particular, TMAC (also referred to as TMAC tolerance). As used herein, the term “high TMAC tolerance” refers to the ability of a DNA polymerase to substantially maintain its enzymatic activity at a TMAC (tetra-methyl ammonium chloride) concentration higher than 10 mM (e.g., higher than 15 mM, 20 mM). Certain characteristics of exemplary DNA polymerases are shown in Table 1.
TABLE 1
Characteristics of exemplary DNA polymerases
Elongation
Fidelity/
Processivity
rate
Salt
Polymerases
Error rate
(nts)
(nts/s)
tolerance
Pfu
2.0 × 10−6
>20
25
30 mM
KOD
4.45 × 10−6
~300
106-138
TNA1
150
T. zilligii
2.0 × 10−6
P. abyssi
0.66 × 10−6
T. gorgonarius
2.2-3.4 × 10−6
Typically, enzymes with high salt tolerance are also characterized with high processivity and/or elongation rate. Without wishing to be bound by any theories, it is thought that salt tolerance affects the binding affinity between polymerase and DNA which, in turn, affects processivity or elongation rate. Typically, binding of polymerases to DNA involves binding interaction between positively charged amino acid residues and negatively charged DNA. At high salt concentrations, competition from the anions of the salt for the positively charged amino acid residues on the polymerases lead to diminished DNA binding affinity. See, Pavlov et al. (2002) Proc. Natl. Acad. Sci. 99(21): 13510-13515, which is incorporated by reference herein. On the other hand, increasing the contact points between DNA and polymerase may increase the salt resistance of the polymerase as well as the processivity or elongation rate because the additional contact points between DNA and polymerase may increase binding affinity of the polymerase for DNA and decrease the rate of dissociation so that the polymerase will remain associated with DNA longer, which will in turn lead to an increase in processivity. For example, Pavlov et al. added helix-hairpin-helix (HhH) motifs from topoisomerase V to Taq and Pfu. These motifs are involved in DNA binding in topoisomerase V. Pavlov et al. showed that both Pfu and Taq become more salt resistant when fused to the HhH motifs. Pavlov et al. also showed that HhH fusion to both Taq and Pfu increased the processivity of the polymerases. As another example, dsDNA binding proteins, e.g., Sso7d, can be fused to DNA polymerases to increase the number of contact points between DNA and polymerases (Wang et al. (2004) Nucl. Acids Res. 32(3): 1197-1207, which is incorporated by reference herein). Sso7d is a sequence non-specific dsDNA binding protein involved in ensuring DNA stability and/or DNA packing in Sulfolobus solfataricus. Fusion of Sso7d to both Taq and Pfu increased the salt resistance and processivity of the polymerases.
Exemplary DNA polymerases characterized with high processivity, elongation rate, thermostability, salt or PCR enhancer tolerance include, but are not limited to, KOD polymerase, TNA1 polymerase, Thermococcus sp. 9 degrees N-7, T4, T7, or phi29. Exemplary DNA polymerases characterized with high fidelity include, but are not limited to, polymerases isolated from Pyrococcus furiosus, P. abyssi, T. gorgonarius, T. litoralis, T. zilligii, T. sp. GT, or P. sp. GB-D.
As non-limiting examples, KOD, Pfu, T. gorgonarius, T. zilligii, T. litoralis and Thermococcus sp. 9N-7 polymerases are used to engineer chimeric DNA polymerases (see the Example sections).
Domains of DNA Polymerases
Typically, archaeal DNA polymerases include at least the following domains: N-terminal domain, exonuclease domain (e.g., 3′->5′ exonuclease domain), palm, fingers, and thumb domain (see
Various functions, such as substrate binding, nucleotide transfer, catalytic activity, proofreading, have been assigned to various domains based on the structural-functional analysis of DNA polymerases. It has also been suggested that the domains tightly coordinate with each other to complete the DNA replication process.
For example, the polymerase activity has been associated with palm, fingers and thumb domains. In particular, the palm subdomain is thought to be the catalytic site of the polymerase. The polymerase catalyzes a phosphoryl transfer reaction in which the alpha phosphate of the incoming dNTP undergoes nucleophilic attack from the OH primer terminus. Typically, three carboxylate side chains are important to this active site. These residues may bind two metal ions (Mg++) which may facilitate deprotonation of the OH terminus and formation of a transition state at the alpha phosphate of the dNTP. The thumb domain is believed to interact with the minor grove of the newly synthesized dsDNA and also with the incoming nucleotide. The thumb domain is less conserved but typically has a largely helical structure. The fingers domain may play a role in template fixation and nucleotide specificity. Like the thumb domain, it is likely to interact with the incoming nucleotide. The thumb domain may contain α helices, and/or β strands. It is thought that unbound DNA polymerases form open conformations of the fingers and thumb domains, and when the DNA is bound, the two domains move towards the palm domain to hold the DNA template and primer more tightly and to probe for Watson-Crick base pairing between the incoming nucleotide and the template nucleotide. The presence of a nucleotide that forms a Watson-Crick base pair with the template facilitates formation of an appropriate conformation of the active site of the polymerase and subsequent incorporation of this nucleotide. For review see Hamilton et al. (2001) BioTechniques 31:370-383. It was reported that mutagenesis in the palm/fingers domain may affects the nucleotide selectivity and affinity and mutagenesis in the thumb domain may affect the binding affinity to dsDNA. Important amino acids in the palm, fingers and thumb domain are described in U.S. Application Publication No. 20060281109, which is hereby incorporated by reference.
The uracil read-ahead function has been associated with the N-terminal domain. For example, archaeal family B DNA polymerases are able to recognize unrepaired uracil in a template strand and stall polymerization upstream of the lesion to prevent an A-T mutation. A “pocket” in the N-terminal domains of archaeal DNA polymerases was identified to be positioned to interact with the template strand and provide this uracil read-ahead function (Fogg et al. (2002) Nature Structural Biology 9(12), 922-927).
The exonuclease domain is associated with either 5′->3′ exonuclease activity, 3′->5″ exonuclease activity or both, which is required to remove incorrectly inserted nucleotide. When a mismatched nucleotide is incorporated, the template/primer strand binds to the polymerase more weakly and/or is misaligned with respect to the polymerase active site causing the mismatched nucleotide to be moved to the active site of the exonuclease domain and excised.
It is thought that the fidelity is affected by the ratio of the polymerase and the exonuclease activity, which may be influenced by the rate of dissociation, conformational change, and the rate of nucleotide incorporation in the presence of mismatched nucleotides. It has also been suggested that the balance between the 3′->5′ exonuclease activity and the polymerase activity is mediated by a flexible loop containing the Y-GG/A motif located between the N-terminal and exonuclease domains and the C-terminal polymerase domains (i.e., the palm, fingers and thumb domains). See, Bohlke et al. (2000) Nucl. Acids Res. 28(20), 3910-3917. A unique loop of the exonuclease domain, and the tip of the thumb are important for the coordination of proofreading and polymerase activities in DNA polymerases. Site-directed mutagenesis in this loop, especially at H147 in KOD DNA polymerase, suggested that electrostatic and hydrophobic interactions between this loop and the thumb affect the ratio between exonuclease activity and polymerase activity and hence fidelity. See, Kuroita et al. J. Mol. Biol. (2005) 351, 291-298.
Domain Swapping
According to the present invention, heterologous domains from different DNA polymerases (e.g., polymerases with at least one distinct functional characteristic) may be combined to form a chimeric polymerase. Suitable domains include naturally-occurring N-terminal domains, exonuclease domains, palm, fingers, and/or thumb domains found in various DNA polymerases. Naturally-occurring N-terminal domains, exonuclease domains, palm, fingers, and/or thumb domains in various DNA polymerases are well defined. For example, an N-terminal domain may include a sequence corresponding to amino acid residues 26 to 105 of KOD polymerase (SEQ ID NO:11); an exonuclease domain may include a region corresponding to amino acid residues 156 to 301 of KOD polymerase (SEQ ID NO:11); a thumb domain may include a region corresponding to amino acid residues 612 to 749 of KOD polymerase (SEQ ID NO:11); and palm and fingers domain may include a region corresponding to amino acid residues 394 to 563 of Pfu polymerase (SEQ ID NO:9).
Corresponding domains or positions in various DNA polymerases can be determined by alignment of amino acid sequences. Alignment of amino acid sequences can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Preferably, the WU-BLAST-2 software is used to determine amino acid sequence identity (Altschul et al., Methods in Enzymology 266, 460-480 (1996); http://blast.wustl/edu/blast/README.html). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11. HSP score (S) and HSP S2 parameters are dynamic values and are established by the program itself, depending upon the composition of the particular sequence, however, the minimum values may be adjusted and are set as indicated above. An example of an alignment is shown in
In some embodiments, a suitable domain may be a variant (e.g., mutant or fragment) of a naturally-occurring domain sequence. For example, a suitable domain may have a sequence having at least 70% (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to an amino acid sequence of a naturally-occurring domain found in a DNA polymerase of interest.
It is further contemplated that sequences defining the N-terminal domain, exonuclease domain, palm, fingers, and/or thumb domains may correlate with certain enzymatic characteristics of DNA polymerases, such as, fidelity or error rate, elongation rate, processivity, and salt resistance. For example, as described in the Examples section, the present inventors have demonstrated that sequences defining the N-terminal, exonuclease, and/or thumb domain may correlate with the characteristics associated with elongation rate, processivity, thermostability, TMAC tolerance and/or salt resistance; and that sequences defining the palm and/or fingers domain may correlate with the characteristics associated with fidelity or error rate of DNA polymerases.
In addition, based on sequence alignments between various DNA polymerases (see, e.g.,
Positive Consensus Sequence 1 (Defining an N-Terminal Domain)
Additionally or alternatively, a domain or domains correlative with high processivity, elongation rate, thermostability, TMAC tolerance and/or salt resistance may be defined by one or more of the following negative consensus sequences:
Negative Consensus Sequence 1 (Defining an N-Terminal Domain)
In some embodiments, a domain correlative with high fidelity may be defined by the following positive consensus sequence (defining palm and fingers domain):
Additionally or alternatively, a domain correlative with high fidelity may be defined by the following negative consensus sequence (defining palm and fingers domain):
Therefore, appropriate domains may be taken or derived from DNA polymerases with distinct functional characteristics to engineer a chimeric DNA polymerase with desirable combinations of functional features. In some embodiments, inventive methods in accordance with the present invention include steps of: (a) providing an N-terminal domain, an exonuclease domain, and/or a thumb domain based on a first DNA polymerase; (b) providing palm and/or fingers domain based on a second DNA polymerase; (c) combining the domains from step (a) and step (b) to form a chimeric polymerase. In some embodiments, the first and the second DNA polymerases are characterized with at least one distinct characteristic. For example, the first DNA polymerase may be characterized with high processivity, elongation rate, thermostability, TMAC tolerance and/or salt resistance and the second DNA polymerase may be characterized with high fidelity. In some embodiments, the first DNA polymerase may be characterized with high fidelity and the second DNA polymerase may be characterized with high processivity, elongation rate, thermostability, TMAC tolerance and/or salt resistance. In some embodiments, a chimeric polymerase engineered according to the invention has a processivity, elongation rate, thermostability, TMAC tolerance or salt resistance substantially similar to that of the first DNA polymerase and a fidelity substantially similar to that of the second DNA polymerase. In some embodiments, a chimeric polymerases engineered according to the present invention has the fidelity higher than that of the first DNA polymerase and the processivity, elongation rate or salt resistance higher than that of the second DNA polymerase.
The present invention further contemplates methods of improving the fidelity, processivity, elongation rate, thermostability, TMAC tolerance and/or salt resistance of a DNA polymerase. In some embodiments, inventive methods in accordance with the invention include a step of replacing a sequence within the palm-fingers domain of the DNA polymerase of interest with a corresponding sequence from a different DNA polymerase that is characterized with higher fidelity relative to the DNA polymerase of interest.
Additionally or alternatively, in some embodiments, inventive methods in accordance with the present invention include a step of replacing a sequence within the N-terminal domain, the exonuclease domain and/or the thumb domain of the DNA polymerase of interest with a corresponding sequence from a different DNA polymerase that is characterized with higher processivity, elongation rate, thermostability, TMAC tolerance or salt resistance relative to the DNA polymerase of interest.
As a non-limiting example, the present inventors have engineered a chimeric DNA polymerase Kofu and its reciprocal chimera POD based on KOD polymerase and Pfu polymerase (see the Examples section). As discussed in the example section, Kofu contains the N-terminal domain, the exonuclease domain and the thumb domain from KOD polymerase and the palm-fingers domain from Pfu polymerase. The sequence of Kofu polymerase is provided in SEQ ID NO:16. The reciprocal chimera POD contains the N-terminal domain, the exonuclease domain and the thumb domain from Pfu polymerase and the palm-fingers domain from KOD polymerase. The sequence of POD polymerase is provided in SEQ ID NO:15.
As discussed in the examples section, the Kofu chimeric polymerase displays the approximate replication fidelity of Pfu but the elongation speed, processivity, thermostability, TMAC tolerance and PCR performance similar to KOD. Alternatively, the Pod chimeric polymerase displays the approximate replication fidelity of KOD but the elongation speed, processivity, thermostability, TMAC tolerance and PCR performance similar to Pfu.
In some embodiments, the present invention provides variants of Kofu chimeric polymerase that contain an amino acid sequence at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO:16 (Kofu amino acid sequence). In particular embodiments, variants of Kofu chimeric polymerase in accordance with the invention have processivity, elongation rate, thermostability, TMAC tolerance and/or fidelity substantially similar to Kofu.
In some embodiments, variants of Kofu chimeric polymerases in accordance with the present invention are defined by consensus sequence XXXXTXXXXXDXXXXXXIXXXXXXEXXXXYXXXXEXXFXXXXKXXXAXXXXXX XXAXXXXTVXTVKRXXXXQXXXXXRXVEXXXXXFTXXXXXXAXXDXIXXXXXXI XXYXXXXXXXXXXXXXXXXVXXXXDXXXXMXXXXXXXXXXXXXXXAEXXXLX XXXXXXEGXRXXXXXXVXXXXXDXXXTXXXXXXXXXXVVKXXXXXVLIXXXXX NXXXAXXKXXCXXXXXNFALXXXXXXXXXXIXXMXXRFXXXXXXXXXXXXXPX XRXXXXXXXXXXXXXXXXVXXQXXXXXXXEXXTXXXTXXXXXXXXRXXXXX XXVXXXXXXXXXXXXAXXXXXVXXPXXXXXXXXXXXXXXXXXXXXXXXXXXV XXXXSXEXYQXXXXEXXTXXFXXXXXKXXXXXXXXXXXXAXXXXXXXXXXXX XXXXXLXXXXNXXIXXXXXXKXXXXIXXXXXXXXXHXXXXXXXXXTXXXEXQX XXXKIXXXXXXKXXXLXXXXFXXXXXXXKXXXXXXXXXXXXXXXXXKXXELVW XXLXXXFXXXXLXIXXXXLYXXXXXGESXEIXXXXLXXLXXXXAXXXXAXXXXX XXXXXXXXXXXXXKXXXXXXXXXITXXXXXXXXXXXXXXXXXXXXXXXXALX XDXXXXKXXXXXXXXTEXXSKXXVXXXXXVXHXXXXXDXKDXXXTXXXXXXX XRXXXRXXXXRXXTXXSXXXXKXSXRXGDXXXPFDXFXXTXXXXXXXXXXXXX XXXXXEXXXRAXXXXXXXXXXXXXXXXXXSZXXKPXGT (SEQ ID NO:38), wherein X is any amino acid or a peptide bond.
In some embodiments, variants of Kofu chimeric polymerases in accordance with the present invention are defined by consensus sequence
In some embodiments, the present invention provide variants of POD chimeric polymerases that contain an amino acid sequence at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO:15 (Pod amino acid sequence). In particular embodiments, variants of POD chimeric polymerases in accordance with the present invention have processivity, elongation rate, thermostability, TMAC tolerance and/or fidelity substantially similar to POD.
Expression of Chimeric DNA Polymerases of the Invention
Standard recombinant DNA techniques (e.g., restriction enzyme digestion, ligation, PCR) can be used to engineer chimeric DNA polymerases in accordance with the present invention. Methods well known in the art may be applied to express and isolate chimeric DNA polymerases. Many bacterial expression vectors contain sequence elements or combinations of sequence elements allowing high level inducible expression of the protein encoded by a foreign sequence. Expression vectors are commercially available from, for example, Novagen (http://www.emdbiosciences.com/html/NVG/AllTables.html#).
In addition, bacteria expressing an integrated inducible form of the T7 RNA polymerase gene may be transformed with an expression vector bearing a chimeric DNA polymerase gene linked to the T7 promoter. Induction of the T7 RNA polymerase by addition of an appropriate inducer, for example, isopropyl-p-D-thiogalactopyranoside (IPTG) for a lac-inducible promoter, induces the high level expression of the chimeric gene from the T7 promoter.
Appropriate host strains of bacteria may be selected from those available in the art by one of skill in the art. As a non-limiting example, E. coli strain BL-21 is commonly used for expression of exogenous proteins since it is protease deficient relative to other strains of E. coli. For situations in which codon usage for the particular polymerase gene differs from that normally seen in E. coli genes, there are strains of BL-21 that are modified to carry tRNA genes encoding tRNAs with rarer anticodons (for example, argU, ileY, leuW, and proL tRNA genes), allowing high efficiency expression of cloned chimeric genes (several BL21-CODON PLUS™ cell strains carrying rare-codon tRNAs are available from Stratagene, for example). Additionally or alternatively, genes encoding DNA polymerases may be codon optimized to facilitate expression in E. coli. Codon optimized sequences can be chemically synthesized.
There are many methods known to those of skill in the art that are suitable for the purification of a chimeric DNA polymerase of the invention. For example, the method of Lawyer et al. (1993, PCR Meth. & App. 2: 275) is well suited for the isolation of DNA polymerases expressed in E. coli, as it was designed originally for the isolation of Taq polymerase. Alternatively, the method of Kong et al. (1993, J. Biol. Chem. 268: 1965, incorporated herein by reference) may be used, which employs a heat denaturation step to destroy host proteins, and two column purification steps (over DEAE-Sepharose and heparin-Sepharose columns) to isolate highly active and approximately 80% pure DNA polymerase.
Further, DNA polymerase mutants may be isolated by an ammonium sulfate fractionation, followed by Q Sepharose and DNA cellulose columns, or by adsorption of contaminants on a HiTrap Q column, followed by gradient elution from a HiTrap heparin column.
Uses of Chimeric DNA Polymerases of the Invention
Chimeric DNA polymerases of the present invention may be used for any methods involving polynucleotide synthesis. Polynucleotide synthesis methods are well known to a person of ordinary skill in the art and can be found, for example, in Molecular Cloning second edition, Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989). For example, chimeric DNA polymerases of the present invention have a variety of uses in recombinant DNA technology including, but not limited to, labeling of DNA by nick translation, second-strand cDNA synthesis in cDNA cloning, DNA sequencing, and amplifying, detecting, and/or cloning nucleic acid sequences using polymerase chain reaction (PCR).
In some embodiments, the invention provides robust, fast, and accurate enzymes for PCR. PCR refers to an in vitro method for amplifying a specific polynucleotide template sequence. The technique of PCR is described in numerous publications, including, PCR: A Practical Approach, M. J. McPherson, et al., IRL Press (1991), PCR Protocols: A Guide to Methods and Applications, by Innis, et al., Academic Press (1990), and PCR Technology: Principals and Applications for DNA Amplification, H. A. Erlich, Stockton Press (1989). PCR is also described in many U.S. Patents, including U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; 4,889,818; 5,075,216; 5,079,352; 5,104,792; 5,023,171; 5,091,310; and 5,066,584, each of which is herein incorporated by reference.
Chimeric DNA polymerases with higher processivity, elongation rate and/or fidelity are expected to reduce error rate, improve efficiency and success rate of long-range amplification (higher yield, longer targets amplified), and/or reduce the amount of required DNA template.
Various specific PCR amplification applications are available in the art (for reviews, see for example, Erlich, 1999, Rev Immunogenet., 1: 127-34; Prediger 2001, Methods Mol. Biol. 160: 49-63; Jurecic et al., 2000, Curr. Opin. Microbiol. 3: 316-21; Triglia, 2000, Methods Mol. Biol. 130: 79-83; MaClelland et al. , 1994, PCR Methods Appl. 4: S66-81; Abramson and Myers, 1993, Current Opinion in Biotechnology 4: 41-47; each of which is incorporated herein by references).
As non-limiting examples, the present invention can be used in PCR applications including, but are not limited to, i) hot-start PCR which reduces non-specific amplification; ii) touch-down PCR which starts at high annealing temperature, then decreases annealing temperature in steps to reduce non-specific PCR product; iii) nested PCR which synthesizes more reliable product using an outer set of primers and an inner set of primers; iv) inverse PCR for amplification of regions flanking a known sequence. In this method, DNA is digested, the desired fragment is circularized by ligation, then PCR using primer complementary to the known sequence extending outwards; v) AP-PCR (arbitrary primed)/RAPD (random amplified polymorphic DNA). These methods create genomic fingerprints from species with little-known target sequences by amplifying using arbitrary oligonucleotides; vi) RT-PCR which uses RNA-directed DNA polymerase (e.g., reverse transcriptase) to synthesize cDNAs which is then used for PCR. This method is extremely sensitive for detecting the expression of a specific sequence in a tissue or cells. It may also be use to quantify mRNA transcripts; vii) RACE (rapid amplification of cDNA ends). This is used where information about DNA/protein sequence is limited. The method amplifies 3′ or 5′ ends of cDNAs generating fragments of cDNA with only one specific primer each (plus one adaptor primer). Overlapping RACE products can then be combined to produce full length cDNA; viii) DD-PCR (differential display PCR) which is used to identify differentially expressed genes in different tissues. First step in DD-PCR involves RT-PCR, then amplification is performed using short, intentionally nonspecific primers; ix) Multiplex-PCR in which two or more unique targets of DNA sequences in the same specimen are amplified simultaneously. One DNA sequence can be use as control to verify the quality of PCR; x) Q/C-PCR (Quantitative comparative) which uses an internal control DNA sequence (but of different size) which compete with the target DNA (competitive PCR) for the same set of primers; xi) Reclusive PCR which is used to synthesize genes. Oligonucleotides used in this method are complementary to stretches of a gene (>80 bases), alternately to the sense and to the antisense strands with ends overlapping (−20 bases); xii) Asymmetric PCR; xiii) In Situ PCR; xiv) Site-directed PCR Mutagenesis; xv) DOP-PCR that uses partially degenerate primers for whole-genome amplification; xvi) quantitative PCR using SYBR green or oligonucleotide probes to detect amplification; xvii) whole-genome amplification using adaptor-ligated DNA fragment libraries as template, and xviii) error-prone PCR in which conditions are optimized to give an increased number of mutations in the PCR product.
It should be understood that this invention is not limited to any particular amplification system. As other systems are developed, those systems may benefit by practice of this invention.
Kits
The invention also contemplates kit formats which include a package unit having one or more containers containing chimeric DNA polymerases of the invention and compositions thereof. In some embodiments, the present invention provides kits further including containers of various reagents used for polynucleotide synthesis, including synthesis in PCR.
Inventive kits in accordance with the present invention may also contain one or more of the following items: polynucleotide precursors, primers, buffers, instructions, and controls. Kits may include containers of reagents mixed together in suitable proportions for performing the methods in accordance with the invention. Reagent containers preferably contain reagents in unit quantities that obviate measuring steps when performing the subject methods.
The two enzymes we chose to include in this experiment were Pyroccocus furiosus DNA polymerase (Pfu) and Thermococcus Kodarensis (KOD) DNA polymerases. The two enzymes have similar domain structure and have a 79% identity at the amino acid level using blastP alignments (see Table 2). The domain structures of Pfu and KOD are illustrated in
TABLE 2
ClustalW alignment of Pfu and KOD
PFU
1
MILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHG
60
KOD
1
....T.....D......I......E....Y....E..F....K...A........A....
60
PFU
61
KIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRY
120
KOD
61
TV.TVKR....Q.....R.VE.....FT......A..D.I......I..Y..........
120
PFU
121
LIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPY
180
KOD
121
......V....D....M...............AE...L.......EG.R......V....
180
PFU
181
VEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFPYLAKRAEKLGIKLTIGRDGSEPK
240
KOD
181
.D...T..........VVK.....VLI.....N...A..K..C.....NFAL........
240
PFU
241
MQRIGDMTAVEVKGRIHFDLYHVITRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWE
300
KOD
241
I..M..RF.............P..R................V..Q.......E..TT...
300
PFU
301
SGENLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWFLLRK
360
KOD
301
T........R.......V............A.....I..S....................
360
PFU
361
AYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLWENIVYLDFRALYPSIIITHNVS
420
KOD
361
......L.....D.K.LA..~.Q..E.. Y.....R............S...........
419
PFU
421
PDTLNLEGCKNYDIAPQVGHKFCKDIPGFIPSLLGHLLEERQKIKTKMKETQDPIEKILL
480
KOD
420
.....R....E..V......R....F.........D.........K...A.I....RK..
479
PFU
481
DYRQKAIKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYIELVWKELEEKFGFKVLYI
540
KOD
480
....R...I....Y.......R.................E..TMTI..I...Y....I.S
539
PFU
541
DTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDE
600
KOD
540
....FF.....ADA.TV....M..L....A....A..................K......
599
PFU
601
EGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDVEEAVRIVKEVIQKLANYEIPPEK
660
KOD
600
...IT........................AL..D....K........TE..SK..V....
659
PFU
661
LAIYEQITRPLHEYKAIGPHVAVAKKLAAKGVKIKPGMVIGYIVLRGDGPISNRAILAEE
720
KOD
660
.V.H.....D.KD...T........R...R....R..T..S....K.S.R.GD...PFD.
719
PFU
721
YDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRYQKTRQVGLTSWLNIKKS*
776
KOD
720
F..T..................E...RA..................SA..KP.GT*
775
PFU
(SEQ ID NO: 9)
KOD
(SEQ ID NO: 11)
Pfu and KOD have very distinct phenotypic characteristics, in particular, with respect to elongation rate, processivity and error rate (See Table 3):
TABLE 3
Pfu
KOD
Elongation Rate:
25 nt/s
106-138 nt/s (Takagi et al. 1997)
Processivity:
>20 nt
~300 nt (Takagi et al. 1997)
Error Rate
1.5 × 10−6
4.45 × 10−6 (internal data)
(mutations/nt/doubling):
Thus, the goal was to find chimeric combinations of these two enzymes which exhibited the error rate comparable to Pfu (2.0×10−6) with the processivity and/or elongation rate comparable to KOD (˜300nt/s and 106-138nt/s respectively). An enzyme with the above mentioned characteristics has utility as a robust, fast, and accurate enzyme for PCR.
Restriction sites were inserted into the codon-optimized nucleotide sequence of KOD and Pfu polymerases at positions that approximately flank the polymerase domain of the enzymes (see Example 2). For example, PvuII and EcoRI sites flanking the polymerase domain (the palm and fingers domain) were used to replace the polymerase domain of Pfu with that of KOD to generate the chimera deemed Pod (
Native DNA sequences for Pyrococcus furiosus polymerase I (SEQ ID NO:1) and Thermococcus kodakarensis polymerase I (SEQ ID NO:2) were retrieved from Genbank. These two DNA sequences were in silico codon optimized by Codon Devices (Cambridge, Mass.) for expression in E. Coli resulting in SEQ ID NO:3 for the Pfu polymerase I codon optimized gene DNA sequence and SEQ ID NO:4 for the KOD polymerase I codon optimized gene DNA sequence. The two codon optimized genes were chemically synthesized and cloned into pUC19 by Codon Devices (Cambridge, Mass.) resulting in SEQ ID NO:7 for Pfu polymerase I and SEQ ID NO:8 for KOD polymerase I.
KOD (SEQ ID NO:8) and Pfu (SEQ ID NO:7) polymerase codon optimized pUC 19 constructs were cloned into the pKBexp vector as follows:
The pKBexp vector contains two Eco31I sites with non-complementary overhangs enabling directional cloning of inserts using a single restriction enzyme. KOD and Pfu polymerase genes were designed with two flanking Eco31I sites that enabled directional and in-frame cloning into pKBexp.
Purified DNA from the pKBexp vector was digested with Eco31I and purified from an agarose gel. KOD and Pfu codon optimized pUC DNA constructs (SEQ ID NO.8 and SEQ ID NO.7) were likewise digested with Eco31I and the roughly 2.3 kilobase insert fragments were cut out from an agarose gel and purified. 30 ng of KOD or Pfu polymerase genes were ligated with 15 ng of digested pKBexp using T4 DNA ligase. The ligation reactions were purified and used to transform competent E. coli DH10B. DNA minipreps were made of ampicillin resistant clones. The presence of inserts was confirmed by digestion of the minipreps with XbaI and HindIII, two enzymes that flank the insert. The cloning of the KOD polymerase gene sequence in pKBexp deemed pKB11 and the Pfu polymerase gene in pKBexp deemed pKB14 were confirmed by DNA sequencing.
The codon-optimized sequences of KOD (SEQ ID NO:5) and Pfu (SEQ ID NO:3) polymerase I genes were designed with restriction sites that approximately flank the finger and palm domains of KOD and Pfu polymerases. The KOD codon optimized sequence contains a PvuII restriction site and an EcoRI restriction site. The Pfu codon optimized sequence contains a PvuII restriction site and an EcoRI restriction site.
Purified DNA from pKB11 and pKB14 were each digested the restriction enzymes EcoRI and PvuII. The large fragment (4.7 kb) and small fragment (0.7 kb) from each digest were separately extracted and purified from an agarose gel. The small fragments from each restriction digest contained the finger and palm domains of KOD and Pfu respectively. The digested and purified large fragments (containing the expression vector and remaining polymerase fragments) were dephosphorylated using Shrimp Alkaline Phosphate. The construct deemed POD was created by ligation of 30 ng of the 4.7 kb Pfu large fragment (aa residues 1 to 335 and 567 to 778 of Pfu DNA polymerase with 10 ng of the 0.7 kb KOD small fragment (corresponding to amino acid residues 336 to 565 of KOD DNA polymerase SEQ ID NO:11). POD thus includes N-terminal, exonuclease and thumb domains from Pfu DNA polymerase and palm and finger domains from KOD. The construct deemed Kofu was made by ligation of 30 ng of the 4.7 kb KOD large fragment (corresponding to amino acid residues 1 to 335 and 566 to 777 of KOD DNA polymerase SEQ ID NO:11) with 10 ng of the 0.7 kb Pfu small fragment (corresponding to amino acid residues 336 to 566 of Pfu DNA polymerase SEQ ID NO:11). Kofu thus includes N-terminal, exonuclease and thumb domains from KOD DNA polymerase and palm and finger domains from Pfu. The ligation reactions were used to transform E. coli DH10B. The construction of Pod (SEQ ID NO:13) and Kofu (SEQ ID NO:14) was confirmed by DNA sequencing. The domain structures of POD and Kofu are illustrated in
10 ng of each enzyme was incubated at 98° C. for 240, 120, 60, 30, 15, 8, 4, 2, 1 or 0 min in a 10 μl volume containing the following: 20 mM Tris-HCl pH 8.0, 2 mM MgCl2, 6 mM (NH4)2SO4, 25 or 50 mM KCl (25 mM for Pfu and Pod, 50 mM for KOD and Kofu). 10 μl of primer/template mix was added to each tube after the heat incubation. The primer template mix contained the following: 20 mM Tris-HCl pH 8.0, 2 mM MgCl2, 6 mM (NH4)2SO4, 0.6 mM dNTP, 0.6 04 each of primers HPRT1-F1 (5′-tttggaaacatctggagtcct -3′ (SEQ ID NO:40)) and HPRT1-R1 (5′-gcccaaagggaactgatagtc -3′ (SEQ ID NO:41)), 2 ng human genomic DNA per μl, and 25 or 50 mM KCl (25 mM for Pfu and Pod, 50 mM for KOD and Kofu). The amplifications were performed with the following cycling protocol: 3 minutes at 95° C., 35×(20 seconds at 98° C., 20 seconds at 60° C., 20 seconds at 72° C.), 20 seconds at 72° C. The PCR products were analysed on an agarose gel (see
The fidelity of enzymes was determined by a method similar to that described by Cline et al. and references therein (Nucl. Acids Res., 1996, 24(18): 3546-3551). LacI was PCR amplified from E. coli and cloned into pUC19 to degenerate plasmid pKB-LacIQZalpha (SEQ ID NO:17). pKB-LacIQZalpha served both as template for PCR amplification of LacI in the fidelity assays and as vector for cloning the amplified LacI into for blue/white colony screening.
2×50 μl PCR reactions (for each enzyme) were set-up, using 70 ng of pKB-LacIQZalfa plasmid template (equivalent to 25 ng of lad target) and 2.5U of each enzyme to amplify the 1.386 Kb lacIOZalpha fragment. The PCR conditions were as follows: amplification with Pfu and Pod were done in Pfu buffer (Fermentas); KOD and Kofu in Novagen KOD buffer 1. Final concentrations of 2 mM MgCl2, 0.4 μM each of primers M13-40 (GTTTTCCCAGTCACGAC (SEQ ID NO:42)) and PKBLac-1R (GGTATCTTTATAGTCCTGTCG (SEQ ID NO:43)) and 0.2 mM each dNTP. Cycling parameters for Pfu and Pod were: 94° C. 4 minutes, 30×(94° C. 15 seconds, 55° C. 15 seconds, 72° C. 3 minutes), 72° C. 6 minutes. Cycling parameters for KOD and Kofu were: 94° C. 2 minutes, 30×(98° C. 15 seconds, 55° C. 2 seconds, 72° C. 20 seconds), 72° C. 30 seconds.
PCR product yields were quantitated by means of gel electrophoresis and the number of template doublings were calculated. PCR products were digested with XbaI, NcoI and DpnI, gel-purified (without exposure to UV light) and ligated into XbaI-NcoI-digested pKB-LacIQZalpha. E. coli was transformed with the ligation mixtures and the cells were plated onto LB-Amp-X-gal plates. The number of blue colonies, white colonies and total number of colonies were recorded. The error rate f was calculated as f=−ln(F)/(d×(bp)), where F=fraction of white colonies ((total colonies minus blue colonies)/total colonies), d=number of template doublings and b=349 (only 349 bp of the lad amplicon are scored). Exemplary results are summarized in Table 4. As shown in Table 4, Pfu and Kofu have similar fidelity and that their fidelity is higher than that of KOD and Pod.
TABLE 4
Fidelity of KOD, Pfu, Kofu and Pod
White
Blue
Total
Fidelity
colonies
Doublings d
colonies
colonies
f (×10−6)
KOD
21130
7.77
246
21376
4.27
Pfu
19270
7.76
77
19347
1.47
Kofu
12817
5.8
39
12856
1.50
Pod
22039
7.19
221
22260
3.98
Processivity can be determined and calculated using assays described in (Wang et al. Nucl. Acids Res, 2004, 32(3): 1197-1207; and Von Hippel et al. NY Acad Sci 1994; 726:118-131). Briefly, 0.8 pmoles of a 5′FAM-labelled primer (−40M13LFF, 5′FAM-GTTTTCCCAGTCACGACGTTGTAAAACGACGGCC-3′ (SEQ ID NO:44)) is added to 1.6 pmoles of ssM13mp18 DNA in the presence of 20 mM Tris-HCl pH 8.0, 25 mM KCl, 2.5 mM MgCl2, 0.3 mM dNTP in a 16 microL volume. The primer is annealed to the template by heating to 95° C. for 2 minutes followed by slow cooling to 72° C. in a thermocycler at a rate of 0.1° C/second, incubation for 10 minutes at 72° C. and further cooling at 0.1° C./second to 4° C. The polymerases are diluted in 20 mM Tris-HCl pH 8.0, 25 mM KCl. The primed template and the diluted polymerases are heated to 72° C. and the reaction is started by adding 4 μl diluted polymerase to 16 μl of primed template. The polymerases are diluted to give polymerase:template ratios of 1:10-1:10000. The reactions are terminated after various timepoints by adding EDTA to a final concentration of 10 mM.
The extension reactions are analyzed on an ABI 3130XL Genetic Analyzer. The median product length is determined for each reaction. The median product length is defined as the length of the product at which the total fluorescence intensity of all products up to that length equals 50% of the sum of fluorescence intensities of all detectable products. The traces for those samples where the median product length does not change with a change in polymerase concentration or incubation time are used to calculate the processivity according to Von Hippel et al. (Von Hippel et al. NY Acad Sci 1994; 726:118-131). Each peak (I) with a fluorescence level significantly above background level is integrated to give the fluorescence intensity of that peak (ni). The total fluorescence intensity (nT) is the sum of the fluorescence of all peaks. The integration data are plotted as log(ni/nT) vs n−1, where n is the number of nucleotides incorporated. The data is fitted to the following equation: log(ni/nT)=(n−1)log Pi+log(1−Pi). Pi, the microscopic processivity factor, is defined as the probability of not terminating extension at position i. The average primer extension length is determined from 1/(1−Pi).
Previous studies (Pavlov et al. (2002) Proc Natl Acad Sci. 99(21), 13510-13515; Wang et al. (2004) Nucl Acids Res. 32(3), 1197-1207) have shown that there is a direct correlation between increased tolerance of polymerases to salt and the processivity of polymerases. For all polymerases tested (from family A or family B), it was found that polymerases with increased salt tolerance also have increased processivity. We therefore compared the salt tolerance of our chimeras with that of the parental polymerases as a proxy for processivity.
The protein concentration of the purified KOD, Pfu, Kofu and Pod where determined using a Bioanalyzer 2100 (Agilent, Santa Clara, Calif., USA) with the Protein 230 Kit from the same supplier. The polymerases were tested in real-time PCR with increasing amounts of KCl added. The reactions were performed in a 20 μl volume containing 20 mM Tris-HCl pH 8.0, 6 mM (NH4)2SO4, 2 mM MgCl2, 3% DMSO, 10 ng polymerase, 20 ng human genomic DNA, 0.3 mM each dNTP, 0.25×SYBR Green (Invitrogen, Carlsbad, Calif., USA). A diluted stock 20×SYBR Green in DMSO was made), 0.3 04 forward primer HPRT1-F1 (5′-tttggaaacatctggagtcct-3′ (SEQ ID NO:40)) and 0.3 μM reverse primer HPRT1-R1(5′-gcccaaagggaactgatagtc-3′ (SEQ ID NO:41)). KCl was added to final concentrations of 10, 25, 50, 75, 100 or 125 mM. PCR amplification was performed in a Corbett 6000 HRM real-time thermocycler (Corbett Life Science, Sidney, Australia) with the following cycling protocol: 3 minutes at 95° C., 40 cycles of (10 seconds at 95° C., 20 seconds at 60° C., 20 seconds at 72° C., data acquisition), followed by a melting curve analysis step of: ramp from 72° C. to 95° C. in 1° C. steps, wait for 5 seconds before data acquisition at the end of each step. 8 μl of each sample was analysed on a 1.5% agarose gel. 5 μl of Fermentas GeneRuler™ Mix, cat no. SM0333 (Fermentas, Vilnius, Lithuania) was loaded onto the gel as a DNA marker. Exemplary results are shown in
Tetra-methyl ammonium-containing salts enhance PCR reactions as shown by Kovarova et al. (Kovarova, M. and Draber, P.; Nucl. Acids Res. (2000) 28(13) e70-). One such salt is tetra-methyl ammonium chloride (TMAC). We therefore compared the TMAC tolerance of our chimeras with that of the parental polymerases.
The polymerases were tested in real-time PCR with increasing amounts of TMAC added. The reactions were performed in a 20 μl volume containing 20 mM Tris-HCl pH 8.0, 6 mM (NH4)2SO4, 2 mM MgCl2, 25 mM KCl, 10 ng polymerase, 20 ng human genomic DNA, 0.3 mM each dNTP, 0.25×SYBR Green (Invitrogen, Carlsbad, Calif., USA. A diluted stock 20×SYBR Green in DMSO was made), 0.3 μM forward primer HPRT1-F1 (5′-tttggaaacatctggagtcct-3′ (SEQ ID NO:40)) and 0.3 μM reverse primer HPRT1-R1(5′-gcccaaagggaactgatagtc-3′ (SEQ ID NO:41)). TMAC was added to final concentrations of 0, 10, 20, 40, 60, 80, 100 or 120 mM. PCR amplification was performed in a Corbett 6000 HRM real-time thermocycler (Corbett Life Science, Sidney, Australia) with the following cycling protocol: 3 minutes at 95° C., 40 cycles of (10 seconds at 95° C., 20 seconds at 50° C., 20 seconds at 72° C., data acquisition), followed by a melting curve analysis step of: ramp from 72° C. to 95° C. in 1° C. steps, wait for 5 seconds before data acquisition at the end of each step. 8 μl of each sample was analysed on a 1.5% agarose gel. 5 μl of Fermentas GeneRuler™ Mix, cat no. SM0333 (Fermentas, Vilnius, Lithuania) was loaded onto the gel as a DNA marker. Exemplary results are shown in
This example is designed to show that the positions where the swapping between domains take place may vary.
Additional chimeras are made by swapping the palm and finger domains of KOD and Pfu polymerases where the exact position of the swap varies slightly compared to positions for Kofu and Pod. Kofu-II (SEQ ID NO:26) is made by replacing amino acid residues 305 to 615 of KOD (SEQ ID NO: 12) with amino acids 305 to 616 of Pfu (SEQ ID NO:10). Pod-II (SEQ ID NO:27) is made by replacing amino acids 305 to 616 of Pfu (SEQ ID NO:10) with amino acids 305 to 615 of KOD (SEQ ID NO:12).
Kofu-III (SEQ ID NO:28) is made by replacing amino acid residues 396 to 564 of KOD (SEQ ID NO: 12) with amino acids 397 to 565 of Pfu (SEQ ID NO:10). Pod-III (SEQ ID NO:29) is made by replacing amino acids 397 to 565 of Pfu (SEQ ID NO:10) with amino acids 396 to 564 of KOD (SEQ ID NO:12).
The amino acid sequence of chimeras Kofu-II, Pod-II, Kofu-III and Pod-III are reverse translated and codon-optimized for expression in E. coli. Additional nucleotide sequences containing Eco31I restriction sites are added to the 5′ and 3′ ends of the construct to facilitate cloning into an expression vector. More specifically, the 5′ and 3′ sequences can be designed so that the overhangs, after digestion of the DNA with Eco31I, are complementary to the overhangs in a particular expression vector (e.g., pKB). Codon optimization and gene synthesis is performed by GeneArt Gmbh. Expression and purification of chimeric polymerases are done using methods known in the art, for example, as reviewed in “Detailed description of the invention”. The thermostability, fidelity, processivity, salt resistance and TMAC resistance of the chimeric polymerases are determined as described in Examples 5 through 9.
Chimeras 9Nli and Li9N are designed based on the alignment in
Chimera 9Nli can be made by replacing the palm and finger region of the 9N polymerase with the palm and finger region of the T. litoralis polymerase. In this particular example, 9Nli is made by replacing amino acids 347 to 580 of 9N polymerase (SEQ ID NO:18) with amino acids 349 to 583 of T. litoralis polymerase (SEQ ID NO:19). The sequence of the coding region of 9Nli is provided as SEQ ID NO:20.
Chimera LiN9 can be made by replacing the palm and finger domain of the DNA polymerase of T. litoralis with the finger domain of the DNA polymerase of 9 degrees North. In this particular example, LiN9 is made by replacing amino acids 349 to 583 of T. litoralis polymerase (SEQ ID NO:19) with amino acids 347 to 580 of 9 degrees N-7 polymerase (SEQ ID NO:18). The sequence of the coding region of LiN9 is provided as SEQ ID NO:21.
Chimerase GoZi and ZiGo are designed based on the alignment in
Chimera GoZi can be made by replacing the palm and finger region of the T. gorgonarius polymerase with the palm and finger region of the T. zilligii polymerase. In this particular example, GoZi is made by replacing amino acids 391 to 559 of T. gorgonarius polymerase (SEQ ID NO:22) with amino acids 391 to 559 of T. zilligii polymerase (SEQ ID NO:23). The sequence of the resulting chimera GoZi is provided as SEQ ID NO:24.
Chimera ZiGo can be made by replacing the palm and finger domain of the DNA polymerase of T. zilligii with the finger domain of the DNA polymerase of T. gorgonarius. In this particular example, ZiGo is made by replacing amino acids 391 to 559 of T. zilligii polymerase (SEQ ID NO:23) with amino acids 391 to 559 of T. gorgonarius polymerase (SEQ ID NO:22). The sequence of the coding region of ZiGo is provided as SEQ ID NO:25.
TABLE 5
Sequences
Native DNA sequences of Pfu and KOD
Sequence 1 (SEQ ID NO: 1)
>Native Pfu nucleotide sequence from genomic sequence (Acc. No. AE010147)
1
ATGATTTTAG ATGTGGATTA CATAACTGAA GAAGGAAAAC CTGTTATTAG GCTATTCAAA
61
AAAGAGAACG GAAAATTTAA GATAGAGCAT GATAGAACTT TTAGACCATA CATTTACGCT
121
CTTCTCAGGG ATGATTCAAA GATTGAAGAA GTTAAGAAAA TAACGGGGGA AAGGCATGGA
181
AAGATTGTGA GAATTGTTGA TGTAGAGAAG GTTGAGAAAA AGTTTCTCGG CAAGCCTATT
241
ACCGTGTGGA AACTTTATTT GGAACATCCC CAAGATGTTC CCACTATTAG AGAAAAAGTT
301
AGAGAACATC CAGCAGTTGT GGACATCTTC GAATACGATA TTCCATTTGC AAAGAGATAC
361
CTCATCGACA AAGGCCTAAT ACCAATGGAG GGGGAAGAAG AGCTAAAGAT TCTTGCCTTC
421
GATATAGAAA CCCTCTATCA CGAAGGAGAA GAGTTTGGAA AAGGCCCAAT TATAATGATT
481
AGTTATGCAG ATGAAAATGA AGCAAAGGTG ATTACTTGGA AAAACATAGA TCTTCCATAC
541
GTTGAGGTTG TATCAAGCGA GAGAGAGATG ATAAAGAGAT TTCTCAGGAT TATCAGGGAG
601
AAGGATCCTG ACATTATAGT TACTTATAAT GGAGACTCAT TCGACTTCCC ATATTTAGCG
661
AAAAGGGCAG AAAAACTTGG GATTAAATTA ACCATTGGAA GAGATGGAAG CGAGCCCAAG
721
ATGCAGAGAA TAGGCGATAT GACGGCTGTA GAAGTCAAGG GAAGAATACA TTTCGACTTG
781
TATCATGTAA TAACAAGGAC AATAAATCTC CCAACATACA CACTAGAGGC TGTATATGAA
841
GCAATTTTTG GAAAGCCAAA GGAGAAGGTA TACGCCGACG AGATAGCAAA AGCCTGGGAA
901
AGTGGAGAGA ACCTTGAGAG AGTTGCCAAA TACTCGATGG AAGATGCAAA GGCAACTTAT
961
GAACTCGGGA AAGAATTCCT TCCAATGGAA ATTCAGCTTT CAAGATTAGT TGGACAACCT
1021
TTATGGGATG TTTCAAGGTC AAGCACAGGG AACCTTGTAG AGTGGTTCTT ACTTAGGAAA
1081
GCCTACGAAA GAAACGAAGT AGCTCCAAAC AAGCCAAGTG AAGAGGAGTA TCAAAGAAGG
1141
CTCAGGGAGA GCTACACAGG TGGATTCGTT AAAGAGCCAG AAAAGGGGTT GTGGGAAAAC
1201
ATAGTATACC TAGATTTTAG AGCCCTATAT CCCTCGATTA TAATTACCCA CAATGTTTCT
1261
CCCGATACTC TAAATCTTGA GGGATGCAAG AACTATGATA TCGCTCCTCA AGTAGGCCAC
1321
AAGTTCTGCA AGGACATCCC TGGTTTTATA CCAAGTCTCT TGGGACATTT GTTAGAGGAA
1381
AGACAAAAGA TTAAGACAAA AATGAAGGAA ACTCAAGATC CTATAGAAAA AATACTCCTT
1441
GACTATAGAC AAAAAGCGAT AAAACTCTTA GCAAATTCTT TCTACGGATA TTATGGCTAT
1501
GCAAAAGCAA GATGGTACTG TAAGGAGTGT GCTGAGAGCG TTACTGCCTG GGGAAGAAAG
1561
TACATCGAGT TAGTATGGAA GGAGCTCGAA GAAAAGTTTG GATTTAAAGT CCTCTACATT
1621
GACACTGATG GTCTCTATGC AACTATCCCA GGAGGAGAAA GTGAGGAAAT AAAGAAAAAG
1681
GCTCTAGAAT TTGTAAAATA CATAAATTCA AAGCTCCCTG GACTGCTAGA GCTTGAATAT
1741
GAAGGGTTTT ATAAGAGGGG ATTCTTCGTT ACGAAGAAGA GGTATGCAGT AATAGATGAA
1801
GAAGGAAAAG TCATTACTCG TGGTTTAGAG ATAGTTAGGA GAGATTGGAG TGAAATTGCA
1861
AAAGAAACTC AAGCTAGAGT TTTGGAGACA ATACTAAAAC ACGGAGATGT TGAAGAAGCT
1921
GTGAGAATAG TAAAAGAAGT AATACAAAAG CTTGCCAATT ATGAAATTCC ACCAGAGAAG
1981
CTCGCAATAT ATGAGCAGAT AACAAGACCA TTACATGAGT ATAAGGCGAT AGGTCCTCAC
2041
GTAGCTGTTG CAAAGAAACT AGCTGCTAAA GGAGTTAAAA TAAAGCCAGG AATGGTAATT
2101
GGATACATAG TACTTAGAGG CGATGGTCCA ATTAGCAATA GGGCAATTCT AGCTGAGGAA
2161
TACGATCCCA AAAAGCACAA GTATGACGCA GAATATTACA TTGAGAACCA GGTTCTTCCA
2221
GCGGTACTTA GGATATTGGA GGGATTTGGA TACAGAAAGG AAGACCTCAG ATACCAAAAG
2281
ACAAGACAAG TCGGCCTAAC TTCCTGGCTT AACATTAAAA AATCCTAG
Sequence 2 (SEQ ID NO: 2)
>Native KOD nucleotide sequence (from genomic sequence, Acc. no. AP006878)
1
ATGATCCTCG ACACTGACTA CATAACCGAG GATGGAAAGC CTGTCATAAG AATTTTCAAG
61
AAGGAAAACG GCGAGTTTAA GATTGAGTAC GACCGGACTT TTGAACCCTA CTTCTACGCC
121
CTCCTGAAGG ACGATTCTGC CATTGAGGAA GTCAAGAAGA TAACCGCCGA GAGGCACGGG
181
ACGGTTGTAA CGGTTAAGCG GGTTGAAAAG GTTCAGAAGA AGTTCCTCGG GAGACCAGTT
241
GAGGTCTGGA AACTCTACTT TACTCATCCG CAGGACGTCC CAGCGATAAG GGACAAGATA
301
CGAGAGCATC CAGCAGTTAT TGACATCTAC GAGTACGACA TACCCTTCGC CAAGCGCTAC
361
CTCATAGACA AGGGATTAGT GCCAATGGAA GGCGACGAGG AGCTGAAAAT GCTCGCCTTC
421
GACATTGAAA CTCTCTACCA TGAGGGCGAG GAGTTCGCCG AGGGGCCAAT CCTTATGATA
481
AGCTACGCCG ACGAGGAAGG GGCCAGGGTG ATAACTTGGA AGAACGTGGA TCTCCCCTAC
541
GTTGACGTCG TCTCGACGGA GAGGGAGATG ATAAAGCGCT TCCTCCGTGT TGTGAAGGAG
601
AAAGACCCGG ACGTTCTCAT AACCTACAAC GGCGACAACT TCGACTTCGC CTATCTGAAA
661
AAGCGCTGTG AAAAGCTCGG AATAAACTTC GCCCTCGGAA GGGATGGAAG CGAGCCGAAG
721
ATTCAGAGGA TGGGCGACAG GTTTGCCGTC GAAGTGAAGG GACGGATACA CTTCGATCTC
781
TATCCTGTGA TAAGACGGAC GATAAACCTG CCCACATACA CGCTTGAGGC CGTTTATGAA
841
GCCGTCTTCG GTCAGCCGAA GGAGAAGGTT TACGCTGAGG AAATAACCAC AGCCTGGGAA
901
ACCGGCGAGA ACCTTGAGAG AGTCGCCCGC TACTCGATGG AAGATGCGAA GGTCACATAC
961
GAGCTTGGGA AGGAGTTCCT TCCGATGGAG GCCCAGCTTT CTCGCTTAAT CGGCCAGTCC
1021
CTCTGGGACG TCTCCCGCTC CAGCACTGGC AACCTCGTTG AGTGGTTCCT CCTCAGGAAG
1081
GCCTATGAGA GGAATGAGCT GGCCCCGAAC AAGCCCGATG AAAAGGAGCT GGCCAGAAGA
1141
CGGCAGAGCT ATGAAGGAGG CTATGTAAAA GAGCCCGAGA GAGGGTTGTG GGAGAACATA
1201
GTGTACCTAG ATTTTAGATC CCTGTACCCC TCAATCATCA TCACCCACAA CGTCTCGCCG
1261
GATACGCTCA ACAGAGAAGG ATGCAAGGAA TATGACGTTG CCCCACAGGT CGGCCACCGC
1321
TTCTGCAAGG ACTTCCCAGG ATTTATCCCG AGCCTGCTTG GAGACCTCCT AGAGGAGAGG
1381
CAGAAGATAA AGAAGAAGAT GAAGGCCACG ATTGACCCGA TCGAGAGGAA GCTCCTCGAT
1441
TACAGGCAGA GGGCCATCAA GATCCTGGCA AACAGCTACT ACGGTTACTA CGGCTATGCA
1501
AGGGCGCGCT GGTACTGCAA GGAGTGTGCA GAGAGCGTAA CGGCCTGGGG AAGGGAGTAC
1561
ATAACGATGA CCATCAAGGA GATAGAGGAA AAGTACGGCT TTAAGGTAAT CTACAGCGAC
1621
ACCGACGGAT TTTTTGCCAC AATACCTGGA GCCGATGCTG AAACCGTCAA AAAGAAGGCT
1681
ATGGAGTTCC TCAAGTATAT CAACGCCAAA CTTCCGGGCG CGCTTGAGCT CGAGTACGAG
1741
GGCTTCTACA AACGCGGCTT CTTCGTCACG AAGAAGAAGT ATGCGGTGAT AGACGAGGAA
1801
GGCAAGATAA CAACGCGCGG ACTTGAGATT GTGAGGCGTG ACTGGAGCGA GATAGCGAAA
1861
GAGACGCAGG CGAGGGTTCT TGAAGCTTTG CTAAAGGACG GTGACGTCGA GAAGGCCGTG
1921
AGGATAGTCA AAGAAGTTAC CGAAAAGCTG AGCAAGTACG AGGTTCCGCC GGAGAAGCTG
1981
GTGATCCACG AGCAGATAAC GAGGGATTTA AAGGACTACA AGGCAACCGG TCCCCACGTT
2041
GCCGTTGCCA AGAGGTTGGC CGCGAGAGGA GTCAAAATAC GCCCTGGAAC GGTGATAAGC
2101
TACATCGTGC TCAAGGGCTC TGGGAGGATA GGCGACAGGG CGATACCGTT CGACGAGTTC
2161
GACCCGACGA AGCACAAGTA CGACGCCGAG TACTACATTG AGAACCAGGT TCTCCCAGCC
2221
GTTGAGAGAA TTCTGAGAGC CTTCGGTTAC CGCAAGGAAG ACCTGCGCTA CCAGAAGACG
2281
AGACAGGTTG GTTTGAGTGC TTGGCTGAAG CCGAAGGGAA CTTGA
Codon optimized sequences of Pfu and KOD
Sequence 3 (SEQ ID NO: 3)
>Pfu codon optimized nucleotide sequence
1
ATGATTCTGG ATGTGGACTA TATCACCGAA GAGGGCAAAC CGGTTATACG TTTATTTAAG
61
AAAGAGAATG GTAAATTCAA GATCGAGCAT GACCGCACGT TCCGTCCATA CATTTACGCG
121
TTGCTTCGGG ATGATAGCAA AATTGAGGAA GTCAAAAAGA TCACCGGGGA ACGTCATGGA
181
AAAATAGTAA GAATTGTGGA CGTTGAAAAA GTCGAAAAGA AATTTCTGGG CAAACCGATC
241
ACTGTATGGA AGCTCTATCT GGAACATCCT CAGGATGTGC CCACAATTCG AGAAAAAGTT
301
CGTGAGCACC CAGCCGTCGT GGATATATTT GAATATGACA TCCCTTTTGC AAAACGCTAC
361
TTAATTGATA AAGGCCTGAT CCCGATGGAG GGGGAAGAAG AACTTAAAAT TCTGGCTTTT
421
GACATAGAAA CGCTCTATCA TGAGGGAGAA GAATTTGGCA AAGGTCCCAT CATTATGATT
481
TCTTACGCGG ATGAGAACGA AGCCAAGGTA ATCACTTGGA AAAATATTGA CCTGCCGTAC
541
GTTGAAGTGG TCAGTTCAGA GCGGGAAATG ATTAAACGTT TTTTACGCAT CATTAGAGAG
601
AAAGATCCAG ATATAATCGT TACATATAAC GGCGACTCCT TCGATTTTCC TTACCTGGCA
661
AAACGAGCTG AAAAATTGGG TATTAAACTT ACCATCGGGC GTGACGGATC GGAACCGAAA
721
ATGCAACGCA TTGGCGATAT GACGGCGGTA GAGGTGAAAG GTCGGATACA CTTTGATCTG
781
TATCATGTCA TCACCCGTAC TATTAATCTC CCCACATACA CGTTAGAAGC CGTTTATGAG
841
GCAATATTCG GCAAGCCGAA AGAAAAAGTG TACGCTGACG AAATCGCGAA GGCATGGGAG
901
AGCGGCGAAA ACCTGGAGCG CGTAGCAAAA TATTCTATGG AAGATGCTAA AGCGACCTAC
961
GAATTGGGGA AAGAATTTCT TCCAATGGAA ATTCAGCTGA GTCGTTTAGT CGGACAACCT
1021
CTGTGGGACG TTTCACGCTC CTCGACTGGC AATCTCGTGG AGTGGTTCCT GTTGAGAAAA
1081
GCCTATGAAC GAAACGAAGT AGCACCGAAT AAACCAAGCG AGGAAGAATA TCAGCGTCGC
1141
CTTCGCGAGT CTTACACAGG TGGGTTTGTT AAGGAACCGG AGAAAGGTCT TTGGGAAAAC
1201
ATCGTGTATT TAGATTTCCG TGCGCTGTAC CCCAGTATTA TAATCACCCA CAATGTCTCA
1261
CCTGACACGC TCAACTTGGA AGGTTGCAAA AATTATGATA TTGCTCCGCA AGTTGGACAT
1321
AAGTTTTGTA AAGATATTCC GGGCTTCATC CCGTCCCTGC TTGGTCACTT ACTGGAAGAG
1381
CGCCAAAAAA TTAAGACCAA AATGAAAGAG ACTCAGGATC CCATTGAAAA GATCCTGCTC
1441
GATTACCGGC AAAAAGCCAT TAAATTGCTT GCAAACTCGT TTTATGGGTA CTATGGCTAT
1501
GCGAAGGCTC GTTGGTACTG CAAAGAATGT GCCGAGAGCG TGACAGCATG GGGTCGCAAA
1561
TATATAGAAT TAGTATGGAA GGAGCTGGAA GAAAAATTCG GATTCAAAGT CCTGTACATC
1621
GATACGGATG GCCTCTATGC GACCATTCCT GGTGGGGAGT CTGAAGAAAT CAAGAAAAAA
1681
GCCTTGGAAT TCGTTAAGTA CATTAATAGT AAATTACCGG GACTGCTTGA ACTGGAGTAT
1741
GAAGGCTTCT ACAAAAGAGG TTTTTTCGTT ACTAAGAAAC GATATGCCGT AATAGATGAA
1801
GAGGGGAAAG TCATCACACG TGGCCTCGAG ATTGTTCGCC GGGACTGGTC AGAGATAGCA
1861
AAGGAAACGC AGGCGCGCGT GCTCGAAACC ATCTTGAAAC ATGGTGATGT AGAGGAAGCC
1921
GTCCGCATTG TTAAAGAGGT GATCCAGAAG TTAGCAAACT ATGAAATTCC ACCGGAAAAA
1981
CTGGCGATAT ACGAGCAAAT CACTCGTCCC CTTCACGAAT ATAAAGCTAT TGGACCTCAT
2041
GTAGCCGTCG CGAAGAAACT GGCTGCAAAA GGCGTTAAGA TAAAACCAGG TATGGTGATC
2101
GGGTACATTG TACTCCGCGG CGACGGTCCG ATTTCCAATA GAGCCATCTT GGCGGAGGAA
2161
TATGATCCTA AAAAGCATAA ATACGACGCT GAATATTACA TTGAGAACCA GGTCTTGCCG
2221
GCAGTTCTGC GGATACTTGA AGGATTTGGC TATCGTAAAG AAGATCTGCG CTATCAAAAG
2281
ACGCGACAGG TGGGTCTGAC TAGCTGGTTG AATATCAAAA AATCGTAA
Sequence 4 (SEQ ID NO: 4)
>Pfu codon optimized nucleotide sequence, extra 9 nt in 5′ area.
1
ATGGCTAGCG CCATTCTGGA TGTGGACTAT ATCACCGAAG AGGGCAAACC GGTTATACGT
61
TTATTTAAGA AAGAGAATGG TAAATTCAAG ATCGAGCATG ACCGCACGTT CCGTCCATAC
121
ATTTACGCGT TGCTTCGGGA TGATAGCAAA ATTGAGGAAG TCAAAAAGAT CACCGGGGAA
181
CGTCATGGAA AAATAGTAAG AATTGTGGAC GTTGAAAAAG TCGAAAAGAA ATTTCTGGGC
241
AAACCGATCA CTGTATGGAA GCTCTATCTG GAACATCCTC AGGATGTGCC CACAATTCGA
301
GAAAAAGTTC GTGAGCACCC AGCCGTCGTG GATATATTTG AATATGACAT CCCTTTTGCA
361
AAACGCTACT TAATTGATAA AGGCCTGATC CCGATGGAGG GGGAAGAAGA ACTTAAAATT
421
CTGGCTTTTG ACATAGAAAC GCTCTATCAT GAGGGAGAAG AATTTGGCAA AGGTCCCATC
481
ATTATGATTT CTTACGCGGA TGAGAACGAA GCCAAGGTAA TCACTTGGAA AAATATTGAC
541
CTGCCGTACG TTGAAGTGGT CAGTTCAGAG CGGGAAATGA TTAAACGTTT TTTACGCATC
601
ATTAGAGAGA AAGATCCAGA TATAATCGTT ACATATAACG GCGACTCCTT CGATTTTCCT
661
TACCTGGCAA AACGAGCTGA AAAATTGGGT ATTAAACTTA CCATCGGGCG TGACGGATCG
721
GAACCGAAAA TGCAACGCAT TGGCGATATG ACGGCGGTAG AGGTGAAAGG TCGGATACAC
781
TTTGATCTGT ATCATGTCAT CACCCGTACT ATTAATCTCC CCACATACAC GTTAGAAGCC
841
GTTTATGAGG CAATATTCGG CAAGCCGAAA GAAAAAGTGT ACGCTGACGA AATCGCGAAG
901
GCATGGGAGA GCGGCGAAAA CCTGGAGCGC GTAGCAAAAT ATTCTATGGA AGATGCTAAA
961
GCGACCTACG AATTGGGGAA AGAATTTCTT CCAATGGAAA TTCAGCTGAG TCGTTTAGTC
1021
GGACAACCTC TGTGGGACGT TTCACGCTCC TCGACTGGCA ATCTCGTGGA GTGGTTCCTG
1081
TTGAGAAAAG CCTATGAACG AAACGAAGTA GCACCGAATA AACCAAGCGA GGAAGAATAT
1141
CAGCGTCGCC TTCGCGAGTC TTACACAGGT GGGTTTGTTA AGGAACCGGA GAAAGGTCTT
1201
TGGGAAAACA TCGTGTATTT AGATTTCCGT GCGCTGTACC CCAGTATTAT AATCACCCAC
1261
AATGTCTCAC CTGACACGCT CAACTTGGAA GGTTGCAAAA ATTATGATAT TGCTCCGCAA
1321
GTTGGACATA AGTTTTGTAA AGATATTCCG GGCTTCATCC CGTCCCTGCT TGGTCACTTA
1381
CTGGAAGAGC GCCAAAAAAT TAAGACCAAA ATGAAAGAGA CTCAGGATCC CATTGAAAAG
1441
ATCCTGCTCG ATTACCGGCA AAAAGCCATT AAATTGCTTG CAAACTCGTT TTATGGGTAC
1501
TATGGCTATG CGAAGGCTCG TTGGTACTGC AAAGAATGTG CCGAGAGCGT GACAGCATGG
1561
GGTCGCAAAT ATATAGAATT AGTATGGAAG GAGCTGGAAG AAAAATTCGG ATTCAAAGTC
1621
CTGTACATCG ATACGGATGG CCTCTATGCG ACCATTCCTG GTGGGGAGTC TGAAGAAATC
1681
AAGAAAAAAG CCTTGGAATT CGTTAAGTAC ATTAATAGTA AATTACCGGG ACTGCTTGAA
1741
CTGGAGTATG AAGGCTTCTA CAAAAGAGGT TTTTTCGTTA CTAAGAAACG ATATGCCGTA
1801
ATAGATGAAG AGGGGAAAGT CATCACACGT GGCCTCGAGA TTGTTCGCCG GGACTGGTCA
1861
GAGATAGCAA AGGAAACGCA GGCGCGCGTG CTCGAAACCA TCTTGAAACA TGGTGATGTA
1921
GAGGAAGCCG TCCGCATTGT TAAAGAGGTG ATCCAGAAGT TAGCAAACTA TGAAATTCCA
1981
CCGGAAAAAC TGGCGATATA CGAGCAAATC ACTCGTCCCC TTCACGAATA TAAAGCTATT
2041
GGACCTCATG TAGCCGTCGC GAAGAAACTG GCTGCAAAAG GCGTTAAGAT AAAACCAGGT
2101
ATGGTGATCG GGTACATTGT ACTCCGCGGC GACGGTCCGA TTTCCAATAG AGCCATCTTG
2161
GCGGAGGAAT ATGATCCTAA AAAGCATAAA TACGACGCTG AATATTACAT TGAGAACCAG
2221
GTCTTGCCGG CAGTTCTGCG GATACTTGAA GGATTTGGCT ATCGTAAAGA AGATCTGCGC
2281
TATCAAAAGA CGCGACAGGT GGGTCTGACT AGCTGGTTGA ATATCAAAAA ATCGTAA
Sequence 5 (SEQ ID NO: 5)
>KOD codon optimized nucleotide sequence
1
ATGATTCTGG ATACCGACTA TATCACGGAA GATGGCAAAC CGGTGATACG TATTTTTAAG
61
AAAGAGAATG GTGAGTTCAA AATCGAGTAC GACCGCACTT TTGAGCCATA TTTCTACGCG
121
TTACTGAAGG ACGATAGCGC CATTGAAGAA GTTAAAAAAA TCACCGCAGA GCGGCATGGG
181
ACAGTGGTAA CCGTGAAGAG AGTTGAAAAA GTCCAGAAAA AATTTTTGGG ACGACCTGTA
241
GAAGTGTGGA AACTTTATTT CACTCACCCC CAAGATGTTC CGGCTATACG TGATAAAATT
301
CGCGAACATC CAGCGGTCAT TGATATTTAC GAATATGATA TACCTTTTGC CAAGCGTTAC
361
CTCATCGACA AAGGCCTGGT GCCGATGGAA GGTGATGAAG AATTAAAAAT GTTGGCATTC
421
GACATTGAAA CACTTTATCA CGAGGGGGAA GAGTTTGCTG AGGGTCCCAT CCTGATGATT
481
TCTTATGCGG ATGAAGAGGG TGCCCGCGTA ATAACCTGGA AGAACGTTGA TCTCCCGTAC
541
GTGGACGTCG TTAGTACGGA ACGGGAAATG ATCAAACGTT TCCTGCGCGT AGTGAAAGAG
601
AAAGATCCAG ACGTCTTAAT TACCTATAAT GGTGATAACT TTGATTTTGC ATACCTGAAA
661
AAAAGATGCG AAAAGTTGGG CATAAATTTC GCTCTTGGTC GAGACGGGTC AGAGCCTAAA
721
ATCCAGCGTA TGGGAGATCG CTTTGCGGTT GAAGTGAAAG GCCGGATTCA TTTCGACCTG
781
TATCCGGTAA TTCGTCGCAC TATCAACCTC CCCACATACA CGTTAGAAGC CGTCTATGAG
841
GCAGTTTTTG GTCAACCGAA GGAAAAAGTT TACGCTGAGG AAATTACCAC TGCGTGGGAA
901
ACAGGCGAGA ATCTGGAACG TGTAGCCCGC TATTCTATGG AGGATGCAAA AGTTACCTAT
961
GAATTGGGTA AGGAATTTCT TCCAATGGAG GCGCAGCTGT CGAGATTAAT AGGGCAGAGC
1021
CTGTGGGACG TGTCTCGAAG TTCAACGGGA AACCTCGTCG AATGGTTTCT GTTGCGGAAA
1081
GCATACGAGC GTAATGAACT TGCCCCTAAC AAACCGGATG AAAAGGAGCT GGCACGCCGT
1141
CGCCAATCCT ATGAAGGCGG TTACGTTAAA GAACCAGAGC GGGGGTTATG GGAAAATATC
1201
GTGTATCTGG ATTTCCGTTC GCTCTACCCG AGCATTATCA TTACCCACAA CGTATCTCCC
1261
GACACTTTGA ATCGCGAGGG CTGTAAAGAA TATGATGTCG CGCCGCAGGT TGGTCATAGA
1321
TTTTGCAAGG ACTTCCCGGG ATTTATACCA AGTCTGCTTG GCGATTTACT GGAAGAGCGA
1381
CAAAAAATCA AAAAGAAAAT GAAAGCTACA ATCGATCCGA TAGAACGTAA GCTGCTCGAC
1441
TACCGCCAGC GGGCCATCAA AATTTTGGCA AACTCATATT ATGGTTACTA TGGGTACGCG
1501
CGTGCTCGCT GGTATTGTAA AGAGTGCGCC GAATCCGTGA CGGCATGGGG CCGTGAATAC
1561
ATCACCATGA CTATTAAGGA GATAGAAGAG AAATATGGTT TCAAAGTAAT CTACTCGGAT
1621
ACAGACGGAT TCTTTGCGAC GATTCCCGGT GCCGATGCAG AAACCGTCAA GAAAAAAGCG
1681
ATGGAATTCC TTAAGTATAT AAATGCTAAA TTACCTGGTG CCCTGGAGCT GGAATACGAA
1741
GGGTTTTACA AACGCGGATT CTTTGTTACT AAGAAAAAAT ATGCGGTGAT CGACGAGGAA
1801
GGCAAGATTA CGACCAGAGG CCTCGAGATT GTACGGCGTG ATTGGAGCGA AATCGCTAAA
1861
GAAACACAGG CACGTGTCTT GGAGGCATTA CTGAAAGATG GGGACGTTGA AAAGGCGGTG
1921
CGAATTGTAA AAGAAGTCAC CGAAAAACTT TCTAAGTACG AAGTTCCGCC AGAGAAACTG
1981
GTGATACACG AACAAATCAC TCGTGATCTG AAAGACTATA AGGCTACAGG CCCGCATGTA
2041
GCAGTCGCCA AACGCCTCGC GGCTCGGGGT GTTAAAATTC GTCCCGGAAC GGTGATCAGT
2101
TACATTGTAT TGAAGGGCTC AGGTCGCATA GGGGATAGAG CAATCCCTTT CGACGAGTTT
2161
GATCCAACCA AACACAAATA TGATGCCGAA TACTATATTG AAAACCAGGT CTTGCCGGCG
2221
GTTGAGCGTA TACTGCGCGC TTTCGGCTAT CGAAAGGAAG ATCTTCGTTA CCAAAAAACT
2281
AGACAGGTGG GTCTGTCCGC ATGGCTCAAA CCTAAGGGAA CGTAA
Sequence 6 (SEQ ID NO: 6)
>KOD codon optimized nucleotide sequence, extra 9 nt in 5′ area.
1
ATGGCTAGCG CCATTCTGGA TACCGACTAT ATCACGGAAG ATGGCAAACC GGTGATACGT
61
ATTTTTAAGA AAGAGAATGG TGAGTTCAAA ATCGAGTACG ACCGCACTTT TGAGCCATAT
121
TTCTACGCGT TACTGAAGGA CGATAGCGCC ATTGAAGAAG TTAAAAAAAT CACCGCAGAG
181
CGGCATGGGA CAGTGGTAAC CGTGAAGAGA GTTGAAAAAG TCCAGAAAAA ATTTTTGGGA
241
CGACCTGTAG AAGTGTGGAA ACTTTATTTC ACTCACCCCC AAGATGTTCC GGCTATACGT
301
GATAAAATTC GCGAACATCC AGCGGTCATT GATATTTACG AATATGATAT ACCTTTTGCC
361
AAGCGTTACC TCATCGACAA AGGCCTGGTG CCGATGGAAG GTGATGAAGA ATTAAAAATG
421
TTGGCATTCG ACATTGAAAC ACTTTATCAC GAGGGGGAAG AGTTTGCTGA GGGTCCCATC
481
CTGATGATTT CTTATGCGGA TGAAGAGGGT GCCCGCGTAA TAACCTGGAA GAACGTTGAT
541
CTCCCGTACG TGGACGTCGT TAGTACGGAA CGGGAAATGA TCAAACGTTT CCTGCGCGTA
601
GTGAAAGAGA AAGATCCAGA CGTCTTAATT ACCTATAATG GTGATAACTT TGATTTTGCA
661
TACCTGAAAA AAAGATGCGA AAAGTTGGGC ATAAATTTCG CTCTTGGTCG AGACGGGTCA
721
GAGCCTAAAA TCCAGCGTAT GGGAGATCGC TTTGCGGTTG AAGTGAAAGG CCGGATTCAT
781
TTCGACCTGT ATCCGGTAAT TCGTCGCACT ATCAACCTCC CCACATACAC GTTAGAAGCC
841
GTCTATGAGG CAGTTTTTGG TCAACCGAAG GAAAAAGTTT ACGCTGAGGA AATTACCACT
901
GCGTGGGAAA CAGGCGAGAA TCTGGAACGT GTAGCCCGCT ATTCTATGGA GGATGCAAAA
961
GTTACCTATG AATTGGGTAA GGAATTTCTT CCAATGGAGG CGCAGCTGTC GAGATTAATA
1021
GGGCAGAGCC TGTGGGACGT GTCTCGAAGT TCAACGGGAA ACCTCGTCGA ATGGTTTCTG
1081
TTGCGGAAAG CATACGAGCG TAATGAACTT GCCCCTAACA AACCGGATGA AAAGGAGCTG
1141
GCACGCCGTC GCCAATCCTA TGAAGGCGGT TACGTTAAAG AACCAGAGCG GGGGTTATGG
1201
GAAAATATCG TGTATCTGGA TTTCCGTTCG CTCTACCCGA GCATTATCAT TACCCACAAC
1261
GTATCTCCCG ACACTTTGAA TCGCGAGGGC TGTAAAGAAT ATGATGTCGC GCCGCAGGTT
1321
GGTCATAGAT TTTGCAAGGA CTTCCCGGGA TTTATACCAA GTCTGCTTGG CGATTTACTG
1381
GAAGAGCGAC AAAAAATCAA AAAGAAAATG AAAGCTACAA TCGATCCGAT AGAACGTAAG
1441
CTGCTCGACT ACCGCCAGCG GGCCATCAAA ATTTTGGCAA ACTCATATTA TGGTTACTAT
1501
GGGTACGCGC GTGCTCGCTG GTATTGTAAA GAGTGCGCCG AATCCGTGAC GGCATGGGGC
1561
CGTGAATACA TCACCATGAC TATTAAGGAG ATAGAAGAGA AATATGGTTT CAAAGTAATC
1621
TACTCGGATA CAGACGGATT CTTTGCGACG ATTCCCGGTG CCGATGCAGA AACCGTCAAG
1681
AAAAAAGCGA TGGAATTCCT TAAGTATATA AATGCTAAAT TACCTGGTGC CCTGGAGCTG
1741
GAATACGAAG GGTTTTACAA ACGCGGATTC TTTGTTACTA AGAAAAAATA TGCGGTGATC
1801
GACGAGGAAG GCAAGATTAC GACCAGAGGC CTCGAGATTG TACGGCGTGA TTGGAGCGAA
1861
ATCGCTAAAG AAACACAGGC ACGTGTCTTG GAGGCATTAC TGAAAGATGG GGACGTTGAA
1921
AAGGCGGTGC GAATTGTAAA AGAAGTCACC GAAAAACTTT CTAAGTACGA AGTTCCGCCA
1981
GAGAAACTGG TGATACACGA ACAAATCACT CGTGATCTGA AAGACTATAA GGCTACAGGC
2041
CCGCATGTAG CAGTCGCCAA ACGCCTCGCG GCTCGGGGTG TTAAAATTCG TCCCGGAACG
2101
GTGATCAGTT ACATTGTATT GAAGGGCTCA GGTCGCATAG GGGATAGAGC AATCCCTTTC
2161
GACGAGTTTG ATCCAACCAA ACACAAATAT GATGCCGAAT ACTATATTGA AAACCAGGTC
2221
TTGCCGGCGG TTGAGCGTAT ACTGCGCGCT TTCGGCTATC GAAAGGAAGA TCTTCGTTAC
2281
CAAAAAACTA GACAGGTGGG TCTGTCCGCA TGGCTCAAAC CTAAGGGAAC GTAA
Sequence 7 (SEQ ID NO: 7)
>pKB13-Pfu codon optimized nucleotide sequence in pUC19 vector
1
TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA
61
CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG
121
TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC
181
ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC
241
ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT
301
TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGT
361
TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT CGGTCTCAGC GCCATTCTGG
421
ATACCGACTA TATCACGGAA GATGGCAAAC CGGTGATACG TATTTTTAAG AAAGAGAATG
481
GTGAGTTCAA AATCGAGTAC GACCGCACTT TTGAGCCATA TTTCTACGCG TTACTGAAGG
541
ACGATAGCGC CATTGAAGAA GTTAAAAAAA TCACCGCAGA GCGGCATGGG ACAGTGGTAA
601
CCGTGAAGAG AGTTGAAAAA GTCCAGAAAA AATTTTTGGG ACGACCTGTA GAAGTGTGGA
661
AACTTTATTT CACTCACCCC CAAGATGTTC CGGCTATACG TGATAAAATT CGCGAACATC
721
CAGCGGTCAT TGATATTTAC GAATATGATA TACCTTTTGC CAAGCGTTAC CTCATCGACA
781
AAGGCCTGGT GCCGATGGAA GGTGATGAAG AATTAAAAAT GTTGGCATTC GACATTGAAA
841
CACTTTATCA CGAGGGGGAA GAGTTTGCTG AGGGTCCCAT CCTGATGATT TCTTATGCGG
901
ATGAAGAGGG TGCCCGCGTA ATAACCTGGA AGAACGTTGA TCTCCCGTAC GTGGACGTCG
961
TTAGTACGGA ACGGGAAATG ATCAAACGTT TCCTGCGCGT AGTGAAAGAG AAAGATCCAG
1021
ACGTCTTAAT TACCTATAAT GGTGATAACT TTGATTTTGC ATACCTGAAA AAAAGATGCG
1081
AAAAGTTGGG CATAAATTTC GCTCTTGGTC GAGACGGGTC AGAGCCTAAA ATCCAGCGTA
1141
TGGGAGATCG CTTTGCGGTT GAAGTGAAAG GCCGGATTCA TTTCGACCTG TATCCGGTAA
1201
TTCGTCGCAC TATCAACCTC CCCACATACA CGTTAGAAGC CGTCTATGAG GCAGTTTTTG
1261
GTCAACCGAA GGAAAAAGTT TACGCTGAGG AAATTACCAC TGCGTGGGAA ACAGGCGAGA
1321
ATCTGGAACG TGTAGCCCGC TATTCTATGG AGGATGCAAA AGTTACCTAT GAATTGGGTA
1381
AGGAATTTCT TCCAATGGAG GCGCAGCTGT CGAGATTAAT AGGGCAGAGC CTGTGGGACG
1441
TGTCTCGAAG TTCAACGGGA AACCTCGTCG AATGGTTTCT GTTGCGGAAA GCATACGAGC
1501
GTAATGAACT TGCCCCTAAC AAACCGGATG AAAAGGAGCT GGCACGCCGT CGCCAATCCT
1561
ATGAAGGCGG TTACGTTAAA GAACCAGAGC GGGGGTTATG GGAAAATATC GTGTATCTGG
1621
ATTTCCGTTC GCTCTACCCG AGCATTATCA TTACCCACAA CGTATCTCCC GACACTTTGA
1681
ATCGCGAGGG CTGTAAAGAA TATGATGTCG CGCCGCAGGT TGGTCATAGA TTTTGCAAGG
1741
ACTTCCCGGG ATTTATACCA AGTCTGCTTG GCGATTTACT GGAAGAGCGA CAAAAAATCA
1801
AAAAGAAAAT GAAAGCTACA ATCGATCCGA TAGAACGTAA GCTGCTCGAC TACCGCCAGC
1861
GGGCCATCAA AATTTTGGCA AACTCATATT ATGGTTACTA TGGGTACGCG CGTGCTCGCT
1921
GGTATTGTAA AGAGTGCGCC GAATCCGTGA CGGCATGGGG CCGTGAATAC ATCACCATGA
1981
CTATTAAGGA GATAGAAGAG AAATATGGTT TCAAAGTAAT CTACTCGGAT ACAGACGGAT
2041
TCTTTGCGAC GATTCCCGGT GCCGATGCAG AAACCGTCAA GAAAAAAGCG ATGGAATTCC
2101
TTAAGTATAT AAATGCTAAA TTACCTGGTG CCCTGGAGCT GGAATACGAA GGGTTTTACA
2161
AACGCGGATT CTTTGTTACT AAGAAAAAAT ATGCGGTGAT CGACGAGGAA GGCAAGATTA
2221
CGACCAGAGG CCTCGAGATT GTACGGCGTG ATTGGAGCGA AATCGCTAAA GAAACACAGG
2281
CACGTGTCTT GGAGGCATTA CTGAAAGATG GGGACGTTGA AAAGGCGGTG CGAATTGTAA
2341
AAGAAGTCAC CGAAAAACTT TCTAAGTACG AAGTTCCGCC AGAGAAACTG GTGATACACG
2401
AACAAATCAC TCGTGATCTG AAAGACTATA AGGCTACAGG CCCGCATGTA GCAGTCGCCA
2461
AACGCCTCGC GGCTCGGGGT GTTAAAATTC GTCCCGGAAC GGTGATCAGT TACATTGTAT
2521
TGAAGGGCTC AGGTCGCATA GGGGATAGAG CAATCCCTTT CGACGAGTTT GATCCAACCA
2581
AACACAAATA TGATGCCGAA TACTATATTG AAAACCAGGT CTTGCCGGCG GTTGAGCGTA
2641
TACTGCGCGC TTTCGGCTAT CGAAAGGAAG ATCTTCGTTA CCAAAAAACT AGACAGGTGG
2701
GTCTGTCCGC ATGGCTCAAA CCTAAGGGAA CGTAATGATA TGAGACCGGA TCCTCTAGAG
2761
TCGACCTGCA GGCATGCAAG CTTGGCGTAA TCATGGTCAT AGCTGTTTCC TGTGTGAAAT
2821
TGTTATCCGC TCACAATTCC ACACAACATA CGAGCCGGAA GCATAAAGTG TAAAGCCTGG
2881
GGTGCCTAAT GAGTGAGCTA ACTCACATTA ATTGCGTTGC GCTCACTGCC CGCTTTCCAG
2941
TCGGGAAACC TGTCGTGCCA GCTGCATTAA TGAATCGGCC AACGCGCGGG GAGAGGCGGT
3001
TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTCACTGACT CGCTGCGCTC GGTCGTTCGG
3061
CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC GGTTATCCAC AGAATCAGGG
3121
GATAACGCAG GAAAGAACAT GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG
3181
GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CGCCCCCCTG ACGAGCATCA CAAAAATCGA
3241
CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC GTTTCCCCCT
3301
GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC TTACCGGATA CCTGTCCGCC
3361
TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCAC GCTGTAGGTA TCTCAGTTCG
3421
GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACGAAC CCCCCGTTCA GCCCGACCGC
3481
TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG TAAGACACGA CTTATCGCCA
3541
CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT ATGTAGGCGG TGCTACAGAG
3601
TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGAA CAGTATTTGG TATCTGCGCT
3661
CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCT CTTGATCCGG CAAACAAACC
3721
ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AAGCAGCAGA TTACGCGCAG AAAAAAAGGA
3781
TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA
3841
CGTTAAGGGA TTTTGGTCAT GAGATTATCA AAAAGGATCT TCACCTAGAT CCTTTTAAAT
3901
TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT AAACTTGGTC TGACAGTTAC
3961
CAATGCTTAA TCAGTGAGGC ACCTATCTCA GCGATCTGTC TATTTCGTTC ATCCATAGTT
4021
GCCTGACTCC CCGTCGTGTA GATAACTACG ATACGGGAGG GCTTACCATC TGGCCCCAGT
4081
GCTGCAATGA TACCGCGAGA CCCACGCTCA CCGGCTCCAG ATTTATCAGC AATAAACCAG
4141
CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT CCTGCAACTT TATCCGCCTC CATCCAGTCT
4201
ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG TTAATAGTTT GCGCAACGTT
4261
GTTGCCATTG CTACAGGCAT CGTGGTGTCA CGCTCGTCGT TTGGTATGGC TTCATTCAGC
4321
TCCGGTTCCC AACGATCAAG GCGAGTTACA TGATCCCCCA TGTTGTGCAA AAAAGCGGTT
4381
AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG CCGCAGTGTT ATCACTCATG
4441
GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT CCGTAAGATG CTTTTCTGTG
4501
ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA TGCGGCGACC GAGTTGCTCT
4561
TGCCCGGCGT CAATACGGGA TAATACCGCG CCACATAGCA GAACTTTAAA AGTGCTCATC
4621
ATTGGAAAAC GTTCTTCGGG GCGAAAACTC TCAAGGATCT TACCGCTGTT GAGATCCAGT
4681
TCGATGTAAC CCACTCGTGC ACCCAACTGA TCTTCAGCAT CTTTTACTTT CACCAGCGTT
4741
TCTGGGTGAG CAAAAACAGG AAGGCAAAAT GCCGCAAAAA AGGGAATAAG GGCGACACGG
4801
AAATGTTGAA TACTCATACT CTTCCTTTTT CAATATTATT GAAGCATTTA TCAGGGTTAT
4861
TGTCTCATGA GCGGATACAT ATTTGAATGT ATTTAGAAAA ATAAACAAAT AGGGGTTCCG
4921
CGCACATTTC CCCGAAAAGT GCCACCTGAC GTCTAAGAAA CCATTATTAT CATGACATTA
4981
ACCTATAAAA ATAGGCGTAT CACGAGGCCC TTTCGTC
Sequence 8 (SEQ ID NO: 8)
>pKB8-KOD codon optimized nucleotide sequence in pUC19 vector
1
TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA
61
CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG
121
TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC
181
ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC
241
ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT
301
TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGT
361
TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT CGGTCTCAGC GCCATTCTGG
421
ATACCGACTA TATCACGGAA GATGGCAAAC CGGTGATACG TATTTTTAAG AAAGAGAATG
481
GTGAGTTCAA AATCGAGTAC GACCGCACTT TTGAGCCATA TTTCTACGCG TTACTGAAGG
541
ACGATAGCGC CATTGAAGAA GTTAAAAAAA TCACCGCAGA GCGGCATGGG ACAGTGGTAA
601
CCGTGAAGAG AGTTGAAAAA GTCCAGAAAA AATTTTTGGG ACGACCTGTA GAAGTGTGGA
661
AACTTTATTT CACTCACCCC CAAGATGTTC CGGCTATACG TGATAAAATT CGCGAACATC
721
CAGCGGTCAT TGATATTTAC GAATATGATA TACCTTTTGC CAAGCGTTAC CTCATCGACA
781
AAGGCCTGGT GCCGATGGAA GGTGATGAAG AATTAAAAAT GTTGGCATTC GACATTGAAA
841
CACTTTATCA CGAGGGGGAA GAGTTTGCTG AGGGTCCCAT CCTGATGATT TCTTATGCGG
901
ATGAAGAGGG TGCCCGCGTA ATAACCTGGA AGAACGTTGA TCTCCCGTAC GTGGACGTCG
961
TTAGTACGGA ACGGGAAATG ATCAAACGTT TCCTGCGCGT AGTGAAAGAG AAAGATCCAG
1021
ACGTCTTAAT TACCTATAAT GGTGATAACT TTGATTTTGC ATACCTGAAA AAAAGATGCG
1081
AAAAGTTGGG CATAAATTTC GCTCTTGGTC GAGACGGGTC AGAGCCTAAA ATCCAGCGTA
1141
TGGGAGATCG CTTTGCGGTT GAAGTGAAAG GCCGGATTCA TTTCGACCTG TATCCGGTAA
1201
TTCGTCGCAC TATCAACCTC CCCACATACA CGTTAGAAGC CGTCTATGAG GCAGTTTTTG
1261
GTCAACCGAA GGAAAAAGTT TACGCTGAGG AAATTACCAC TGCGTGGGAA ACAGGCGAGA
1321
ATCTGGAACG TGTAGCCCGC TATTCTATGG AGGATGCAAA AGTTACCTAT GAATTGGGTA
1381
AGGAATTTCT TCCAATGGAG GCGCAGCTGT CGAGATTAAT AGGGCAGAGC CTGTGGGACG
1441
TGTCTCGAAG TTCAACGGGA AACCTCGTCG AATGGTTTCT GTTGCGGAAA GCATACGAGC
1501
GTAATGAACT TGCCCCTAAC AAACCGGATG AAAAGGAGCT GGCACGCCGT CGCCAATCCT
1561
ATGAAGGCGG TTACGTTAAA GAACCAGAGC GGGGGTTATG GGAAAATATC GTGTATCTGG
1621
ATTTCCGTTC GCTCTACCCG AGCATTATCA TTACCCACAA CGTATCTCCC GACACTTTGA
1681
ATCGCGAGGG CTGTAAAGAA TATGATGTCG CGCCGCAGGT TGGTCATAGA TTTTGCAAGG
1741
ACTTCCCGGG ATTTATACCA AGTCTGCTTG GCGATTTACT GGAAGAGCGA CAAAAAATCA
1801
AAAAGAAAAT GAAAGCTACA ATCGATCCGA TAGAACGTAA GCTGCTCGAC TACCGCCAGC
1861
GGGCCATCAA AATTTTGGCA AACTCATATT ATGGTTACTA TGGGTACGCG CGTGCTCGCT
1921
GGTATTGTAA AGAGTGCGCC GAATCCGTGA CGGCATGGGG CCGTGAATAC ATCACCATGA
1981
CTATTAAGGA GATAGAAGAG AAATATGGTT TCAAAGTAAT CTACTCGGAT ACAGACGGAT
2041
TCTTTGCGAC GATTCCCGGT GCCGATGCAG AAACCGTCAA GAAAAAAGCG ATGGAATTCC
2101
TTAAGTATAT AAATGCTAAA TTACCTGGTG CCCTGGAGCT GGAATACGAA GGGTTTTACA
2161
AACGCGGATT CTTTGTTACT AAGAAAAAAT ATGCGGTGAT CGACGAGGAA GGCAAGATTA
2221
CGACCAGAGG CCTCGAGATT GTACGGCGTG ATTGGAGCGA AATCGCTAAA GAAACACAGG
2281
CACGTGTCTT GGAGGCATTA CTGAAAGATG GGGACGTTGA AAAGGCGGTG CGAATTGTAA
2341
AAGAAGTCAC CGAAAAACTT TCTAAGTACG AAGTTCCGCC AGAGAAACTG GTGATACACG
2401
AACAAATCAC TCGTGATCTG AAAGACTATA AGGCTACAGG CCCGCATGTA GCAGTCGCCA
2461
AACGCCTCGC GGCTCGGGGT GTTAAAATTC GTCCCGGAAC GGTGATCAGT TACATTGTAT
2521
TGAAGGGCTC AGGTCGCATA GGGGATAGAG CAATCCCTTT CGACGAGTTT GATCCAACCA
2581
AACACAAATA TGATGCCGAA TACTATATTG AAAACCAGGT CTTGCCGGCG GTTGAGCGTA
2641
TACTGCGCGC TTTCGGCTAT CGAAAGGAAG ATCTTCGTTA CCAAAAAACT AGACAGGTGG
2701
GTCTGTCCGC ATGGCTCAAA CCTAAGGGAA CGTAATGATA TGAGACCGGA TCCTCTAGAG
2761
TCGACCTGCA GGCATGCAAG CTTGGCGTAA TCATGGTCAT AGCTGTTTCC TGTGTGAAAT
2821
TGTTATCCGC TCACAATTCC ACACAACATA CGAGCCGGAA GCATAAAGTG TAAAGCCTGG
2881
GGTGCCTAAT GAGTGAGCTA ACTCACATTA ATTGCGTTGC GCTCACTGCC CGCTTTCCAG
2941
TCGGGAAACC TGTCGTGCCA GCTGCATTAA TGAATCGGCC AACGCGCGGG GAGAGGCGGT
3001
TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTCACTGACT CGCTGCGCTC GGTCGTTCGG
3061
CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC GGTTATCCAC AGAATCAGGG
3121
GATAACGCAG GAAAGAACAT GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG
3181
GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CGCCCCCCTG ACGAGCATCA CAAAAATCGA
3241
CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC GTTTCCCCCT
3301
GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC TTACCGGATA CCTGTCCGCC
3361
TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCAC GCTGTAGGTA TCTCAGTTCG
3421
GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACGAAC CCCCCGTTCA GCCCGACCGC
3481
TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG TAAGACACGA CTTATCGCCA
3541
CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT ATGTAGGCGG TGCTACAGAG
3601
TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGAA CAGTATTTGG TATCTGCGCT
3661
CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCT CTTGATCCGG CAAACAAACC
3721
ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AAGCAGCAGA TTACGCGCAG AAAAAAAGGA
3781
TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA
3841
CGTTAAGGGA TTTTGGTCAT GAGATTATCA AAAAGGATCT TCACCTAGAT CCTTTTAAAT
3901
TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT AAACTTGGTC TGACAGTTAC
3961
CAATGCTTAA TCAGTGAGGC ACCTATCTCA GCGATCTGTC TATTTCGTTC ATCCATAGTT
4021
GCCTGACTCC CCGTCGTGTA GATAACTACG ATACGGGAGG GCTTACCATC TGGCCCCAGT
4081
GCTGCAATGA TACCGCGAGA CCCACGCTCA CCGGCTCCAG ATTTATCAGC AATAAACCAG
4141
CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT CCTGCAACTT TATCCGCCTC CATCCAGTCT
4201
ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG TTAATAGTTT GCGCAACGTT
4261
GTTGCCATTG CTACAGGCAT CGTGGTGTCA CGCTCGTCGT TTGGTATGGC TTCATTCAGC
4321
TCCGGTTCCC AACGATCAAG GCGAGTTACA TGATCCCCCA TGTTGTGCAA AAAAGCGGTT
4381
AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG CCGCAGTGTT ATCACTCATG
4441
GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT CCGTAAGATG CTTTTCTGTG
4501
ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA TGCGGCGACC GAGTTGCTCT
4561
TGCCCGGCGT CAATACGGGA TAATACCGCG CCACATAGCA GAACTTTAAA AGTGCTCATC
4621
ATTGGAAAAC GTTCTTCGGG GCGAAAACTC TCAAGGATCT TACCGCTGTT GAGATCCAGT
4681
TCGATGTAAC CCACTCGTGC ACCCAACTGA TCTTCAGCAT CTTTTACTTT CACCAGCGTT
4741
TCTGGGTGAG CAAAAACAGG AAGGCAAAAT GCCGCAAAAA AGGGAATAAG GGCGACACGG
4801
AAATGTTGAA TACTCATACT CTTCCTTTTT CAATATTATT GAAGCATTTA TCAGGGTTAT
4861
TGTCTCATGA GCGGATACAT ATTTGAATGT ATTTAGAAAA ATAAACAAAT AGGGGTTCCG
4921
CGCACATTTC CCCGAAAAGT GCCACCTGAC GTCTAAGAAA CCATTATTAT CATGACATTA
4981
ACCTATAAAA ATAGGCGTAT CACGAGGCCC TTTCGTC
Amino acid sequences of Pfu and KOD
Sequence 9 (SEQ ID NO: 9)
>Pfu amino acid sequence
1
MILDVDYITE EGKPVIRLFK KENGKFKIEH DRTFRPYIYA LLRDDSKIEE VKKITGERHG
61
KIVRIVDVEK VEKKFLGKPI TVWKLYLEHP QDVPTIREKV REHPAVVDIF EYDIPFAKRY
121
LIDKGLIPME GEEELKILAF DIETLYHEGE EFGKGPIIMI SYADENEAKV ITWKNIDLPY
181
VEVVSSEREM IKRFLRIIRE KDPDIIVTYN GDSFDFPYLA KRAEKLGIKL TIGRDGSEPK
241
MQRIGDMTAV EVKGRIHFDL YHVITRTINL PTYTLEAVYE AIFGKPKEKV YADEIAKAWE
301
SGENLERVAK YSMEDAKATY ELGKEFLPME IQLSRLVGQP LWDVSRSSTG NLVEWFLLRK
361
AYERNEVAPN KPSEEEYQRR LRESYTGGFV KEPEKGLWEN IVYLDFRALY PSIIITHNVS
421
PDTLNLEGCK NYDIAPQVGH KFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILL
481
DYRQKAIKLL ANSFYGYYGY AKARWYCKEC AESVTAWGRK YIELVWKELE EKFGFKVLYI
541
DTDGLYATIP GGESEEIKKK ALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDE
601
EGKVITRGLE IVRRDWSEIA KETQARVLET ILKHGDVEEA VRIVKEVIQK LANYEIPPEK
661
LAIYEQITRP LHEYKAIGPH VAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEE
721
YDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQK TRQVGLTSWL NIKKS*
Sequence 10 (SEQ ID NO: 10)
>Pfu amino acid sequence, extra 3 aa in 5′ area.
1
MASAILDVDY ITEEGKPVIR LFKKENGKFK IEHDRTFRPY IYALLRDDSK IEEVKKITGE
61
RHGKIVRIVD VEKVEKKFLG KPITVWKLYL EHPQDVPTIR EKVREHPAVV DIFEYDIPFA
121
KRYLIDKGLI PMEGEEELKI LAFDIETLYH EGEEFGKGPI IMISYADENE AKVITWKNID
181
LPYVEVVSSE REMIKRFLRI IREKDPDIIV TYNGDSFDFP YLAKRAEKLG IKLTIGRDGS
241
EPKMQRIGDM TAVEVKGRIH FDLYHVITRT INLPTYTLEA VYEAIFGKPK EKVYADEIAK
301
AWESGENLER VAKYSMEDAK ATYELGKEFL PMEIQLSRLV GQPLWDVSRS STGNLVEWFL
361
LRKAYERNEV APNKPSEEEY QRRLRESYTG GFVKEPEKGL WENIVYLDFR ALYPSIIITH
421
NVSPDTLNLE GCKNYDIAPQ VGHKFCKDIP GFIPSLLGHL LEERQKIKTK MKETQDPIEK
481
ILLDYRQKAI KLLANSFYGY YGYAKARWYC KECAESVTAW GRKYIELVWK ELEEKFGFKV
541
LYIDTDGLYA TIPGGESEEI KKKALEFVKY INSKLPGLLE LEYEGFYKRG FFVTKKRYAV
601
IDEEGKVITR GLEIVRRDWS EIAKETQARV LETILKHGDV EEAVRIVKEV IQKLANYEIP
661
PEKLAIYEQI TRPLHEYKAI GPHVAVAKKL AAKGVKIKPG MVIGYIVLRG DGPISNRAIL
721
AEEYDPKKHK YDAEYYIENQ VLPAVLRILE GFGYRKEDLR YQKTRQVGLT SWLNIKKS*
Sequence 11 (SEQ ID NO: 11)
>KOD amino acid sequence
1
MILDTDYITE DGKPVIRIFK KENGEFKIEY DRTFEPYFYA LLKDDSAIEE VKKITAERHG
61
TVVTVKRVEK VQKKFLGRPV EVWKLYFTHP QDVPAIRDKI REHPAVIDIY EYDIPFAKRY
121
LIDKGLVPME GDEELKMLAF DIETLYHEGE EFAEGPILMI SYADEEGARV ITWKNVDLPY
181
VDVVSTEREM IKRFLRVVKE KDPDVLITYN GDNFDFAYLK KRCEKLGINF ALGRDGSEPK
241
IQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLEAVYE AVFGQPKEKV YAEEITTAWE
301
TGENLERVAR YSMEDAKVTY ELGKEFLPME AQLSRLIGQS LWDVSRSSTG NLVEWFLLRK
361
AYERNELAPN KPDEKELARR RQSYEGGYVK EPERGLWENI VYLDFRSLYP SIIITHNVSP
421
DTLNREGCKE YDVAPQVGHR FCKDFPGFIP SLLGDLLEER QKIKKKMKAT IDPIERKLLD
481
YRQRAIKILA NSYYGYYGYA RARWYCKECA ESVTAWGREY ITMTIKEIEE KYGFKVIYSD
541
TDGFFATIPG ADAETVKKKA MEFLKYINAK LPGALELEYE GFYKRGFFVT KKKYAVIDEE
601
GKITTRGLEI VRRDWSEIAK ETQARVLEAL LKDGDVEKAV RIVKEVTEKL SKYEVPPEKL
661
VIHEQITRDL KDYKATGPHV AVAKRLAARG VKIRPGTVIS YIVLKGSGRI GDRAIPFDEF
721
DPTKHKYDAE YYIENQVLPA VERILRAFGY RKEDLRYQKT RQVGLSAWLK PKGT
Sequence 12 (SEQ ID NO: 12)
>KOD amino acid sequence, extra 3 aa in 5′ area.
1
MASAILDTDY ITEDGKPVIR IFKKENGEFK IEYDRTFEPY FYALLKDDSA IEEVKKITAE
61
RHGTVVTVKR VEKVQKKFLG RPVEVWKLYF THPQDVPAIR DKIREHPAVI DIYEYDIPFA
121
KRYLIDKGLV PMEGDEELKM LAFDIETLYH EGEEFAEGPI LMISYADEEG ARVITWKNVD
181
LPYVDVVSTE REMIKRFLRV VKEKDPDVLI TYNGDNFDFA YLKKRCEKLG INFALGRDGS
241
EPKIQRMGDR FAVEVKGRIH FDLYPVIRRT INLPTYTLEA VYEAVFGQPK EKVYAEEITT
301
AWETGENLER VARYSMEDAK VTYELGKEFL PMEAQLSRLI GQSLWDVSRS STGNLVEWFL
361
LRKAYERNEL APNKPDEKEL ARRRQSYEGG YVKEPERGLW ENIVYLDFRS LYPSIIITHN
421
VSPDTLNREG CKEYDVAPQV GHRFCKDFPG FIPSLLGDLL EERQKIKKKM KATIDPIERK
481
LLDYRQRAIK ILANSYYGYY GYARARWYCK ECAESVTAWG REYITMTIKE IEEKYGFKVI
541
YSDTDGFFAT IPGADAETVK KKAMEFLKYI NAKLPGALEL EYEGFYKRGF FVTKKKYAVI
601
DEEGKITTRG LEIVRRDWSE IAKETQARVL EALLKDGDVE KAVRIVKEVT EKLSKYEVPP
661
EKLVIHEQIT RDLKDYKATG PHVAVAKRLA ARGVKIRPGT VISYIVLKGS GRIGDRAIPF
721
DEFDPTKHKY DAEYYIENQV LPAVERILRA FGYRKEDLRY QKTRQVGLSA WLKPKGT*
DNA sequences of chimeras Pod and Kofu
Sequence 13 (SEQ ID NO: 13)
>Pod codon optimized nucleotide sequence
1
ATGGCTAGCG CCATTCTGGA TGTGGACTAT ATCACCGAAG AGGGCAAACC GGTTATACGT
61
TTATTTAAGA AAGAGAATGG TAAATTCAAG ATCGAGCATG ACCGCACGTT CCGTCCATAC
121
ATTTACGCGT TGCTTCGGGA TGATAGCAAA ATTGAGGAAG TCAAAAAGAT CACCGGGGAA
181
CGTCATGGAA AAATAGTAAG AATTGTGGAC GTTGAAAAAG TCGAAAAGAA ATTTCTGGGC
241
AAACCGATCA CTGTATGGAA GCTCTATCTG GAACATCCTC AGGATGTGCC CACAATTCGA
301
GAAAAAGTTC GTGAGCACCC AGCCGTCGTG GATATATTTG AATATGACAT CCCTTTTGCA
361
AAACGCTACT TAATTGATAA AGGCCTGATC CCGATGGAGG GGGAAGAAGA ACTTAAAATT
421
CTGGCTTTTG ACATAGAAAC GCTCTATCAT GAGGGAGAAG AATTTGGCAA AGGTCCCATC
481
ATTATGATTT CTTACGCGGA TGAGAACGAA GCCAAGGTAA TCACTTGGAA AAATATTGAC
541
CTGCCGTACG TTGAAGTGGT CAGTTCAGAG CGGGAAATGA TTAAACGTTT TTTACGCATC
601
ATTAGAGAGA AAGATCCAGA TATAATCGTT ACATATAACG GCGACTCCTT CGATTTTCCT
661
TACCTGGCAA AACGAGCTGA AAAATTGGGT ATTAAACTTA CCATCGGGCG TGACGGATCG
721
GAACCGAAAA TGCAACGCAT TGGCGATATG ACGGCGGTAG AGGTGAAAGG TCGGATACAC
781
TTTGATCTGT ATCATGTCAT CACCCGTACT ATTAATCTCC CCACATACAC GTTAGAAGCC
841
GTTTATGAGG CAATATTCGG CAAGCCGAAA GAAAAAGTGT ACGCTGACGA AATCGCGAAG
901
GCATGGGAGA GCGGCGAAAA CCTGGAGCGC GTAGCAAAAT ATTCTATGGA AGATGCTAAA
961
GCGACCTACG AATTGGGGAA AGAATTTCTT CCAATGGAAA TTCAGCTGTC GAGATTAATA
1021
GGGCAGAGCC TGTGGGACGT GTCTCGAAGT TCAACGGGAA ACCTCGTCGA ATGGTTTCTG
1081
TTGCGGAAAG CATACGAGCG TAATGAACTT GCCCCTAACA AACCGGATGA AAAGGAGCTG
1141
GCACGCCGTC GCCAATCCTA TGAAGGCGGT TACGTTAAAG AACCAGAGCG GGGGTTATGG
1201
GAAAATATCG TGTATCTGGA TTTCCGTTCG CTCTACCCGA GCATTATCAT TACCCACAAC
1261
GTATCTCCCG ACACTTTGAA TCGCGAGGGC TGTAAAGAAT ATGATGTCGC GCCGCAGGTT
1321
GGTCATAGAT TTTGCAAGGA CTTCCCGGGA TTTATACCAA GTCTGCTTGG CGATTTACTG
1381
GAAGAGCGAC AAAAAATCAA AAAGAAAATG AAAGCTACAA TCGATCCGAT AGAACGTAAG
1441
CTGCTCGACT ACCGCCAGCG GGCCATCAAA ATTTTGGCAA ACTCATATTA TGGTTACTAT
1501
GGGTACGCGC GTGCTCGCTG GTATTGTAAA GAGTGCGCCG AATCCGTGAC GGCATGGGGC
1561
CGTGAATACA TCACCATGAC TATTAAGGAG ATAGAAGAGA AATATGGTTT CAAAGTAATC
1621
TACTCGGATA CAGACGGATT CTTTGCGACG ATTCCCGGTG CCGATGCAGA AACCGTCAAG
1681
AAAAAAGCGA TGGAATTCGT TAAGTACATT AATAGTAAAT TACCGGGACT GCTTGAACTG
1741
GAGTATGAAG GCTTCTACAA AAGAGGTTTT TTCGTTACTA AGAAACGATA TGCCGTAATA
1801
GATGAAGAGG GGAAAGTCAT CACACGTGGC CTCGAGATTG TTCGCCGGGA CTGGTCAGAG
1861
ATAGCAAAGG AAACGCAGGC GCGCGTGCTC GAAACCATCT TGAAACATGG TGATGTAGAG
1921
GAAGCCGTCC GCATTGTTAA AGAGGTGATC CAGAAGTTAG CAAACTATGA AATTCCACCG
1981
GAAAAACTGG CGATATACGA GCAAATCACT CGTCCCCTTC ACGAATATAA AGCTATTGGA
2041
CCTCATGTAG CCGTCGCGAA GAAACTGGCT GCAAAAGGCG TTAAGATAAA ACCAGGTATG
2101
GTGATCGGGT ACATTGTACT CCGCGGCGAC GGTCCGATTT CCAATAGAGC CATCTTGGCG
2161
GAGGAATATG ATCCTAAAAA GCATAAATAC GACGCTGAAT ATTACATTGA GAACCAGGTC
2221
TTGCCGGCAG TTCTGCGGAT ACTTGAAGGA TTTGGCTATC GTAAAGAAGA TCTGCGCTAT
2281
CAAAAGACGC GACAGGTGGG TCTGACTAGC TGGTTGAATA TCAAAAAATC GTAA
Sequence 14 (SEQ ID NO: 14)
>Kofu codon optimized nucleotide sequence
1
ATGGCTAGCG CCATTCTGGA TACCGACTAT ATCACGGAAG ATGGCAAACC GGTGATACGT
61
ATTTTTAAGA AAGAGAATGG TGAGTTCAAA ATCGAGTACG ACCGCACTTT TGAGCCATAT
121
TTCTACGCGT TACTGAAGGA CGATAGCGCC ATTGAAGAAG TTAAAAAAAT CACCGCAGAG
181
CGGCATGGGA CAGTGGTAAC CGTGAAGAGA GTTGAAAAAG TCCAGAAAAA ATTTTTGGGA
241
CGACCTGTAG AAGTGTGGAA ACTTTATTTC ACTCACCCCC AAGATGTTCC GGCTATACGT
301
GATAAAATTC GCGAACATCC AGCGGTCATT GATATTTACG AATATGATAT ACCTTTTGCC
361
AAGCGTTACC TCATCGACAA AGGCCTGGTG CCGATGGAAG GTGATGAAGA ATTAAAAATG
421
TTGGCATTCG ACATTGAAAC ACTTTATCAC GAGGGGGAAG AGTTTGCTGA GGGTCCCATC
481
CTGATGATTT CTTATGCGGA TGAAGAGGGT GCCCGCGTAA TAACCTGGAA GAACGTTGAT
541
CTCCCGTACG TGGACGTCGT TAGTACGGAA CGGGAAATGA TCAAACGTTT CCTGCGCGTA
601
GTGAAAGAGA AAGATCCAGA CGTCTTAATT ACCTATAATG GTGATAACTT TGATTTTGCA
661
TACCTGAAAA AAAGATGCGA AAAGTTGGGC ATAAATTTCG CTCTTGGTCG AGACGGGTCA
721
GAGCCTAAAA TCCAGCGTAT GGGAGATCGC TTTGCGGTTG AAGTGAAAGG CCGGATTCAT
781
TTCGACCTGT ATCCGGTAAT TCGTCGCACT ATCAACCTCC CCACATACAC GTTAGAAGCC
841
GTCTATGAGG CAGTTTTTGG TCAACCGAAG GAAAAAGTTT ACGCTGAGGA AATTACCACT
901
GCGTGGGAAA CAGGCGAGAA TCTGGAACGT GTAGCCCGCT ATTCTATGGA GGATGCAAAA
961
GTTACCTATG AATTGGGTAA GGAATTTCTT CCAATGGAGG CGCAGCTGAG TCGTTTAGTC
1021
GGACAACCTC TGTGGGACGT TTCACGCTCC TCGACTGGCA ATCTCGTGGA GTGGTTCCTG
1081
TTGAGAAAAG CCTATGAACG AAACGAAGTA GCACCGAATA AACCAAGCGA GGAAGAATAT
1141
CAGCGTCGCC TTCGCGAGTC TTACACAGGT GGGTTTGTTA AGGAACCGGA GAAAGGTCTT
1201
TGGGAAAACA TCGTGTATTT AGATTTCCGT GCGCTGTACC CCAGTATTAT AATCACCCAC
1261
AATGTCTCAC CTGACACGCT CAACTTGGAA GGTTGCAAAA ATTATGATAT TGCTCCGCAA
1321
GTTGGACATA AGTTTTGTAA AGATATTCCG GGCTTCATCC CGTCCCTGCT TGGTCACTTA
1381
CTGGAAGAGC GCCAAAAAAT TAAGACCAAA ATGAAAGAGA CTCAGGATCC CATTGAAAAG
1441
ATCCTGCTCG ATTACCGGCA AAAAGCCATT AAATTGCTTG CAAACTCGTT TTATGGGTAC
1501
TATGGCTATG CGAAGGCTCG TTGGTACTGC AAAGAATGTG CCGAGAGCGT GACAGCATGG
1561
GGTCGCAAAT ATATAGAATT AGTATGGAAG GAGCTGGAAG AAAAATTCGG ATTCAAAGTC
1621
CTGTACATCG ATACGGATGG CCTCTATGCG ACCATTCCTG GTGGGGAGTC TGAAGAAATC
1681
AAGAAAAAAG CCTTGGAATT CCTTAAGTAT ATAAATGCTA AATTACCTGG TGCCCTGGAG
1741
CTGGAATACG AAGGGTTTTA CAAACGCGGA TTCTTTGTTA CTAAGAAAAA ATATGCGGTG
1801
ATCGACGAGG AAGGCAAGAT TACGACCAGA GGCCTCGAGA TTGTACGGCG TGATTGGAGC
1861
GAAATCGCTA AAGAAACACA GGCACGTGTC TTGGAGGCAT TACTGAAAGA TGGGGACGTT
1921
GAAAAGGCGG TGCGAATTGT AAAAGAAGTC ACCGAAAAAC TTTCTAAGTA CGAAGTTCCG
1981
CCAGAGAAAC TGGTGATACA CGAACAAATC ACTCGTGATC TGAAAGACTA TAAGGCTACA
2041
GGCCCGCATG TAGCAGTCGC CAAACGCCTC GCGGCTCGGG GTGTTAAAAT TCGTCCCGGA
2101
ACGGTGATCA GTTACATTGT ATTGAAGGGC TCAGGTCGCA TAGGGGATAG AGCAATCCCT
2161
TTCGACGAGT TTGATCCAAC CAAACACAAA TATGATGCCG AATACTATAT TGAAAACCAG
2221
GTCTTGCCGG CGGTTGAGCG TATACTGCGC GCTTTCGGCT ATCGAAAGGA AGATCTTCGT
2281
TACCAAAAAA CTAGACAGGT GGGTCTGTCC GCATGGCTCA AACCTAAGGG AACGTAA
Amino acid sequences of chimeras Pod and Kofu
Sequence 15 (SEQ ID NO: 15)
>Pod amino acid sequence
1
MASAILDVDY ITEEGKPVIR LFKKENGKFK IEHDRTFRPY IYALLRDDSK IEEVKKITGE
61
RHGKIVRIVD VEKVEKKFLG KPITVWKLYL EHPQDVPTIR EKVREHPAVV DIFEYDIPFA
121
KRYLIDKGLI PMEGEEELKI LAFDIETLYH EGEEFGKGPI IMISYADENE AKVITWKNID
181
LPYVEVVSSE REMIKRFLRI IREKDPDIIV TYNGDSFDFP YLAKRAEKLG IKLTIGRDGS
241
EPKMQRIGDM TAVEVKGRIH FDLYHVITRT INLPTYTLEA VYEAIFGKPK EKVYADEIAK
301
AWESGENLER VAKYSMEDAK ATYELGKEFL PMEIQLSRLI GQSLWDVSRS STGNLVEWFL
361
LRKAYERNEL APNKPDEKEL ARRRQSYEGG YVKEPERGLW ENIVYLDFRS LYPSIIITHN
421
VSPDTLNREG CKEYDVAPQV GHRFCKDFPG FIPSLLGDLL EERQKIKKKM KATIDPIERK
481
LLDYRQRAIK ILANSYYGYY GYARARWYCK ECAESVTAWG REYITMTIKE IEEKYGFKVI
541
YSDTDGFFAT IPGADAETVK KKAMEFVKYI NSKLPGLLEL EYEGFYKRGF FVTKKRYAVI
601
DEEGKVITRG LEIVRRDWSE IAKETQARVL ETILKHGDVE EAVRIVKEVI QKLANYEIPP
661
EKLAIYEQIT RPLHEYKAIG PHVAVAKKLA AKGVKIKPGM VIGYIVLRGD GPISNRAILA
721
EEYDPKKHKY DAEYYIENQV LPAVLRILEG FGYRKEDLRY QKTRQVGLTS WLNIKKS*
Sequence 16 (SEQ ID NO: 16)
>Kofu amino acid sequence
1
MASAILDTDY ITEDGKPVIR IFKKENGEFK IEYDRTFEPY FYALLKDDSA IEEVKKITAE
61
RHGTVVTVKR VEKVQKKFLG RPVEVWKLYF THPQDVPAIR DKIREHPAVI DIYEYDIPFA
121
KRYLIDKGLV PMEGDEELKM LAFDIETLYH EGEEFAEGPI LMISYADEEG ARVITWKNVD
181
LPYVDVVSTE REMIKRFLRV VKEKDPDVLI TYNGDNFDFA YLKKRCEKLG INFALGRDGS
241
EPKIQRMGDR FAVEVKGRIH FDLYPVIRRT INLPTYTLEA VYEAVFGQPK EKVYAEEITT
301
AWETGENLER VARYSMEDAK VTYELGKEFL PMEAQLSRLV GQPLWDVSRS STGNLVEWFL
361
LRKAYERNEV APNKPSEEEY QRRLRESYTG GFVKEPEKGL WENIVYLDFR ALYPSIIITH
421
NVSPDTLNLE GCKNYDIAPQ VGHKFCKDIP GFIPSLLGHL LEERQKIKTK MKETQDPIEK
481
ILLDYRQKAI KLLANSFYGY YGYAKARWYC KECAESVTAW GRKYIELVWK ELEEKFGFKV
541
LYIDTDGLYA TIPGGESEEI KKKALEFLKY INAKLPGALE LEYEGFYKRG FFVTKKKYAV
601
IDEEGKITTR GLEIVRRDWS EIAKETQARV LEALLKDGDV EKAVRIVKEV TEKLSKYEVP
661
PEKLVIHEQI TRDLKDYKAT GPHVAVAKRL AARGVKIRPG TVISYIVLKG SGRIGDRAIP
721
FDEFDPTKHK YDAEYYIENQ VLPAVERILR AFGYRKEDLR YQKTRQVGLS AWLKPKGT*
Sequence 17 (SEQ ID NO: 17)
>pLACIQZa
1
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA
61
CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG
121
TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC
181
ACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCC
241
ATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTAT
GT
301
TACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGT
TTTCCCAGTCACGAC >>> Primer M13-40 (SEQ ID NO: 42)
361
TTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCGGTACCCGGGGAT
XbaI
421
CCTCTAGAGCCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACA
481
ATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTG
541
AGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCG
601
TGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGC
661
CAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTG
721
GCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTG
781
TTTGATGGTGGTTGACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCAC
841
TACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAG
901
CGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTG
961
CATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTG
1021
AATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGA
1081
ACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCAC
1141
GCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGA
1201
GACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTG
1261
GTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCAC
1321
CGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACC
1381
CAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAG
1441
ACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCG
1501
GTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGA
1561
AACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTC
1621
TGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGG
1681
GCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCAACGTAAATGCA
NcoI
1741
TGCCGCTTCGCCTTCCGGCCACCAGAATAGCCTGCGCCATGGGCTTCCTCGCTCACTGAC
1801
TCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATA
1861
CGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAA
1921
AAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCT
1981
GACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA
PRIMER PKBLACIR <<< GCTGTCCTGATATT
TCTATGG (SEQ ID NO: 43)
2041
AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCG
2101
CTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCA
2161
CGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAA
2221
CCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCG
2281
GTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGG
2341
TATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGA
2401
ACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGC
2461
TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAG
2521
ATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGAC
2581
GCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATC
2641
TTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAG
2701
TAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGT
2761
CTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAG
2821
GGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA
2881
GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACT
2941
TTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCA
3001
GTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCG
3061
TTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCC
3121
ATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTG
3181
GCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCA
3241
TCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGT
3301
ATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC
3361
AGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATC
3421
TTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCA
3481
TCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAA
3541
AAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTAT
3601
TGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAA
3661
AATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAA
3721
ACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC
Amino acid sequences of DNA polymerases from T. litoralis, Thermococcus sp. 9 degrees
N-7 and chimeras thereof.
Sequence 18 (SEQ ID NO: 18)
Thermococcus sp. 9 degrees N-7 DNA polymerase amino acid sequence (acc no. U47108)
1
MILDTDYITE NGKPVIRVFK KENGEFKIEY DRTFEPYFYA LLKDDSAIED VKKVTAKRHG
61
TVVKVKRAEK VQKKFLGRPI EVWKLYFNHP QDVPAIRDRI RAHPAVVDIY EYDIPFAKRY
121
LIDKGLIPME GDEELTMLAF DIETLYHEGE EFGTGPILMI SYADGSEARV ITWKKIDLPY
181
VDVVSTEKEM IKRFLRVVRE KDPDVLITYN GDNFDFAYLK KRCEELGIKF TLGRDGSEPK
241
IQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLEAVYE AVFGKPKEKV YAEEIAQAWE
301
SGEGLERVAR YSMEDAKVTY ELGREFFPME AQLSRLIGQS LWDVSRSSTG NLVEWFLLRK
361
AYKRNELAPN KPDERELARR RGGYAGGYVK EPERGLWDNI VYLDFRSLYP SIIITHNVSP
421
DTLNREGCKE YDVAPEVGHK FCKDFPGFIP SLLGDLLEER QKIKRKMKAT VDPLEKKLLD
481
YRQRAIKILA NSFYGYYGYA KARWYCKECA ESVTAWGREY IEMVIRELEE KFGFKVLYAD
541
TDGLHATIPG ADAETVKKKA KEFLKYINPK LPGLLELEYE GFYVRGFFVT KKKYAVIDEE
601
GKITTRGLEI VRRDWSEIAK ETQARVLEAI LKHGDVEEAV RIVKEVTEKL SKYEVPPEKL
661
VIHEQITRDL RDYKATGPHV AVAKRLAARG VKIRPGTVIS YIVLKGSGRI GDRAIPADEF
721
DPTKHRYDAE YYIENQVLPA VERILKAFGY RKEDLRYQKT KQVGLGAWLK VKGKK
Sequence 19 (SEQ ID NO: 19)
T. litoralis DNA polymerase amino acid sequence (acc no. M74198.1)
1
MILDTDYITK DGKPIIRIFK KENGEFKIEL DPHFQPYIYA LLKDDSAIEE IKAIKGERHG
61
KTVRVLDAVK VRKKFLGREV EVWKLIFEHP QDVPAMRGKI REHPAVVDIY EYDIPFAKRY
121
LIDKGLIPME GDEELKLLAF DIETFYHEGD EFGKGEIIMI SYADEEEARV ITWKNIDLPY
181
VDVVSNEREM IKRFVQVVKE KDPDVIITYN GDNFDLPYLI KRAEKLGVRL VLGRDKEHPE
241
PKIQRMGDSF AVEIKGRIHF DLFPVVRRTI NLPTYTLEAV YEAVLGKTKS KLGAEEIAAI
301
WETEESMKKL AQYSMEDARA TYELGKEFFP MEAELAKLIG QSVWDVSRSS TGNLVEWYLL
361
RVAYARNELA PNKPDEEEYK RRLRTTYLGG YVKEPEKGLW ENIIYLDFRS LYPSIIVTHN
421
VSPDTLEKEG CKNYDVAPIV GYRFCKDFPG FIPSILGDLI AMRQDIKKKM KSTIDPIEKK
481
MLDYRQRAIK LLANSYYGYM GYPKARWYSK ECAESVTAWG RHYIEMTIRE IEEKFGFKVL
541
YADTDGFYAT IPGEKPELIK KKAKEFLNYI NSKLPGLLEL EYEGFYLRGF FVTKKRYAVI
601
DEEGRITTRG LEVVRRDWSE IAKETQAKVL EAILKEGSVE KAVEVVRDVV EKIAKYRVPL
661
EKLVIHEQIT RDLKDYKAIG PHVAIAKRLA ARGIKVKPGT IISYIVLKGS GKISDRVILL
721
TEYDPRKHKY DPDYYIENQV LPAVLRILEA FGYRKEDLRY QSSKQTGLDA WLKR
Sequence 20 (SEQ ID NO: 20)
Amino acid sequence of chimeric DNA polymerase 9Nli
1
MILDTDYITE NGKPVIRVFK KENGEFKIEY DRTFEPYFYA LLKDDSAIED VKKVTAKRHG
61
TVVKVKRAEK VQKKFLGRPI EVWKLYFNHP QDVPAIRDRI RAHPAVVDIY EYDIPFAKRY
121
LIDKGLIPME GDEELTMLAF DIETLYHEGE EFGTGPILMI SYADGSEARV ITWKKIDLPY
181
VDVVSTEKEM IKRFLRVVRE KDPDVLITYN GDNFDFAYLK KRCEELGIKF TLGRDGSEPK
241
IQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLEAVYE AVFGKPKEKV YAEEIAQAWE
301
SGEGLERVAR YSMEDAKVTY ELGREFFPME AQLSRLIGQS LWDVSRSSTG NLVEWYLLRV
361
AYARNELAPN KPDEEEYKRR LRTTYLGGYV KEPEKGLWEN IIYLDFRSLY PSIIVTHNVS
421
PDTLEKEGCK NYDVAPIVGY RFCKDFPGFI PSILGDLIAM RQDIKKKMKS TIDPIEKKML
481
DYRQRAIKLL ANSYYGYMGY PKARWYSKEC AESVTAWGRH YIEMTIREIE EKFGFKVLYA
541
DTDGFYATIP GEKPELIKKK AKEFLNYINS KLPGLLELEY EGFYVRGFFV TKKKYAVIDE
601
EGKITTRGLE IVRRDWSEIA KETQARVLEA ILKHGDVEEA VRIVKEVTEK LSKYEVPPEK
661
LVIHEQITRD LRDYKATGPH VAVAKRLAAR GVKIRPGTVI SYIVLKGSGR IGDRAIPADE
721
FDPTKHRYDA EYYIENQVLP AVERILKAFG YRKEDLRYQK TKQVGLGAWL KVKGKK
Sequence 21 (SEQ ID NO: 21)
Amino acid sequence of chimeric DNA polymerase Li9N
1
MILDTDYITK DGKPIIRIFK KENGEFKIEL DPHFQPYIYA LLKDDSAIEE IKAIKGERHG
61
KTVRVLDAVK VRKKFLGREV EVWKLIFEHP QDVPAMRGKI REHPAVVDIY EYDIPFAKRY
121
LIDKGLIPME GDEELKLLAF DIETFYHEGD EFGKGEIIMI SYADEEEARV ITWKNIDLPY
181
VDVVSNEREM IKRFVQVVKE KDPDVIITYN GDNFDLPYLI KRAEKLGVRL VLGRDKEHPE
241
PKIQRMGDSF AVEIKGRIHF DLFPVVRRTI NLPTYTLEAV YEAVLGKTKS KLGAEEIAAI
301
WETEESMKKL AQYSMEDARA TYELGKEFFP MEAELAKLIG QSVWDVSRSS TGNLVEWFLL
361
RKAYKRNELA PNKPDERELA RRRGGYAGGY VKEPERGLWD NIVYLDFRSL YPSIIITHNV
421
SPDTLNREGC KEYDVAPEVG HKFCKDFPGF IPSLLGDLLE ERQKIKRKMK ATVDPLEKKL
481
LDYRQRAIKI LANSFYGYYG YAKARWYCKE CAESVTAWGR EYIEMVIREL EEKFGFKVLY
541
ADTDGLHATI PGADAETVKK KAKEFLKYIN PKLPGLLELE YEGFYLRGFF VTKKRYAVID
601
EEGRITTRGL EVVRRDWSEI AKETQAKVLE AILKEGSVEK AVEVVRDVVE KIAKYRVPLE
661
KLVIHEQITR DLKDYKAIGP HVAIAKRLAA RGIKVKPGTI ISYIVLKGSG KISDRVILLT
721
EYDPRKHKYD PDYYIENQVL PAVLRILEAF GYRKEDLRYQ SSKQTGLDAW LKR
Amino acid sequences of DNA polymerases from T. gorgonarius, T. zilligii and chimeras
thereof.
Sequence 22 (SEQ ID NO: 22)
T. gorgonarius DNA polymerase amino acid sequence (acc no. 4699806)
1
MILDTDYITE DGKPVIRIFK KENGEFKIDY DRNFEPYIYA LLKDDSAIED VKKITAERHG
61
TTVRVVRAEK VKKKFLGRPI EVWKLYFTHP QDVPAIRDKI KEHPAVVDIY EYDIPFAKRY
121
LIDKGLIPME GDEELKMLAF DIETLYHEGE EFAEGPILMI SYADEEGARV ITWKNIDLPY
181
VDVVSTEKEM IKRFLKVVKE KDPDVLITYN GDNFDFAYLK KRSEKLGVKF ILGREGSEPK
241
IQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLEAVYE AIFGQPKEKV YAEEIAQAWE
301
TGEGLERVAR YSMEDAKVTY ELGKEFFPME AQLSRLVGQS LWDVSRSSTG NLVEWFLLRK
361
AYERNELAPN KPDERELARR RESYAGGYVK EPERGLWENI VYLDFRSLYP SIIITHNVSP
421
DTLNREGCEE YDVAPQVGHK FCKDFPGFIP SLLGDLLEER QKVKKKMKAT IDPIEKKLLD
481
YRQRAIKILA NSFYGYYGYA KARWYCKECA ESVTAWGRQY IETTIREIEE KFGFKVLYAD
541
TDGFFATIPG ADAETVKKKA KEFLDYINAK LPGLLELEYE GFYKRGFFVT KKKYAVIDEE
601
DKITTRGLEI VRRDWSEIAK ETQARVLEAI LKHGDVEEAV RIVKEVTEKL SKYEVPPEKL
661
VIYEQITRDL KDYKATGPHV AVAKRLAARG IKIRPGTVIS YIVLKGSGRI GDRAIPFDEF
721
DPAKHKYDAE YYIENQVLPA VERILRAFGY RKEDLRYQKT RQVGLGAWLK PKT
Sequence 23 (SEQ ID NO: 23)
T. zilligii DNA polymerase amino acid sequence
1
MILDADYITE DGKPVIRVFK KEKGEFKIDY DRDFEPYIYA LLKDDSAIED IKKITAERHG
61
TTVRVTRAER VKKKFLGRPV EVWKLYFTHP QDVPAIRDKI REHPAVVDIY EYDIPFAKRY
121
LIDRGLIPME GDEELRMLAF DIETLYHEGE EFGEGPILMI SYADEEGARV ITWKNIDLPY
181
VESVSTEKEM IKRFLKVIQE KDPDVLITYN GDNFDFAYLK KRSETLGVKF ILGRDGSEPK
241
IQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLETVYE AIFGQPKEKV YAEEIARAWE
301
SGEGLERVAR YSMEDAKATY ELGKEFFPME AQLSRLVGQS LWDVSRSSTG NLVEWFLLRK
361
AYERNELAPN KPDERELARR AESYAGGYVK EPEKGLWENI VYLDYKSLYP SIIITHNVSP
421
DTLNREGCRE YDVAPQVGHR FCKDFPGFIP SLLGDLLEER QKVKKKMKAT VDPIERKLLD
481
YRQRAIKILA NSYYGYYGYA NARWYCRECA ESVTAWGRQY IETTMREIEE KFGFKVLYAD
541
TDGFFATIPG ADAETVKKKA KEFLNYINPR LPGLLELEYE GFYRRGFFVT KKKYAVIDEE
601
DKITTRGLEI VRRDWSEIAK ETQARVLEAI LKHGDVEEAV RIVKEVTEKL SRYEVPPEKL
661
VIYEQITRDL RDYRATGPHV AVAKRLAARG IKIRPGTVIS YIVLKGPGRV GDRAIPFDEF
721
DPAKHRYDAE YYIENQVLPA VERILRAFGY RKEDLRYQKT KQAGLGAWLK PKT
Sequence 24 (SEQ ID NO: 24)
Amino acid sequence of chimeric DNA polymerase GoZi
1
MILDTDYITE DGKPVIRIFK KENGEFKIDY DRNFEPYIYA LLKDDSAIED VKKITAERHG
61
TTVRVVRAEK VKKKFLGRPI EVWKLYFTHP QDVPAIRDKI KEHPAVVDIY EYDIPFAKRY
121
LIDKGLIPME GDEELKMLAF DIETLYHEGE EFAEGPILMI SYADEEGARV ITWKNIDLPY
181
VDVVSTEKEM IKRFLKVVKE KDPDVLITYN GDNFDFAYLK KRSEKLGVKF ILGREGSEPK
241
IQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLEAVYE AIFGQPKEKV YAEEIAQAWE
301
TGEGLERVAR YSMEDAKVTY ELGKEFFPME AQLSRLVGQS LWDVSRSSTG NLVEWFLLRK
361
AYERNELAPN KPDERELARR RESYAGGYVK EPEKGLWENI VYLDYKSLYP SIIITHNVSP
421
DTLNREGCRE YDVAPQVGHR FCKDFPGFIP SLLGDLLEER QKVKKKMKAT VDPIERKLLD
481
YRQRAIKILA NSYYGYYGYA NARWYCRECA ESVTAWGRQY IETTMREIEE KFGFKVLYAD
541
TDGFFATIPG ADAETVKKKA KEFLDYINAK LPGLLELEYE GFYKRGFFVT KKKYAVIDEE
601
DKITTRGLEI VRRDWSEIAK ETQARVLEAI LKHGDVEEAV RIVKEVTEKL SKYEVPPEKL
661
VIYEQITRDL KDYKATGPHV AVAKRLAARG IKIRPGTVIS YIVLKGSGRI GDRAIPFDEF
721
DPAKHKYDAE YYIENQVLPA VERILRAFGY RKEDLRYQKT RQVGLGAWLK PKT
Sequence 25 (SEQ ID NO: 25)
Amino acid sequence of chimeric DNA polymerase ZiGo
1
MILDADYITE DGKPVIRVFK KEKGEFKIDY DRDFEPYIYA LLKDDSAIED IKKITAERHG
61
TTVRVTRAER VKKKFLGRPV EVWKLYFTHP QDVPAIRDKI REHPAVVDIY EYDIPFAKRY
121
LIDRGLIPME GDEELRMLAF DIETLYHEGE EFGEGPILMI SYADEEGARV ITWKNIDLPY
181
VESVSTEKEM IKRFLKVIQE KDPDVLITYN GDNFDFAYLK KRSETLGVKF ILGRDGSEPK
241
IQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLETVYE AIFGQPKEKV YAEEIARAWE
301
SGEGLERVAR YSMEDAKATY ELGKEFFPME AQLSRLVGQS LWDVSRSSTG NLVEWFLLRK
361
AYERNELAPN KPDERELARR AESYAGGYVK EPERGLWENI VYLDFRSLYP SIIITHNVSP
421
DTLNREGCEE YDVAPQVGHK FCKDFPGFIP SLLGDLLEER QKVKKKMKAT IDPIEKKLLD
481
YRQRAIKILA NSFYGYYGYA KARWYCKECA ESVTAWGRQY IETTIREIEE KFGFKVLYAD
541
TDGFFATIPG ADAETVKKKA KEFLNYINPR LPGLLELEYE GFYRRGFFVT KKKYAVIDEE
601
DKITTRGLEI VRRDWSEIAK ETQARVLEAI LKHGDVEEAV RIVKEVTEKL SRYEVPPEKL
661
VIYEQITRDL RDYRATGPHV AVAKRLAARG IKIRPGTVIS YIVLKGPGRV GDRAIPFDEF
721
DPAKHRYDAE YYIENQVLPA VERILRAFGY RKEDLRYQKT KQAGLGAWLK PKT
Amino acid sequences of additional chimeras of KOD and Pfu DNA polymerases.
Sequence 26 (SEQ ID NO: 26)
Amino acid sequence of chimeric DNA polymerase Kofu-II.
1
MASAILDTDY ITEDGKPVIR IFKKENGEFK IEYDRTFEPY FYALLKDDSA IEEVKKITAE
61
RHGTVVTVKR VEKVQKKFLG RPVEVWKLYF THPQDVPAIR DKIREHPAVI DIYEYDIPFA
121
KRYLIDKGLV PMEGDEELKM LAFDIETLYH EGEEFAEGPI LMISYADEEG ARVITWKNVD
181
LPYVDVVSTE REMIKRFLRV VKEKDPDVLI TYNGDNFDFA YLKKRCEKLG INFALGRDGS
241
EPKIQRMGDR FAVEVKGRIH FDLYPVIRRT INLPTYTLEA VYEAVFGQPK EKVYAEEITT
301
AWETGENLER VAKYSMEDAK ATYELGKEFL PMEIQLSRLV GQPLWDVSRS STGNLVEWFL
361
LRKAYERNEV APNKPSEEEY QRRLRESYTG GFVKEPEKGL WENIVYLDFR ALYPSIIITH
421
NVSPDTLNLE GCKNYDIAPQ VGHKFCKDIP GFIPSLLGHL LEERQKIKTK MKETQDPIEK
481
ILLDYRQKAI KLLANSFYGY YGYAKARWYC KECAESVTAW GRKYIELVWK ELEEKFGFKV
541
LYIDTDGLYA TIPGGESEEI KKKALEFVKY INSKLPGLLE LEYEGFYKRG FFVTKKRYAV
601
IDEEGKVITR GLEIVRRDWS EIAKETQARV LEALLKDGDV EKAVRIVKEV TEKLSKYEVP
661
PEKLVIHEQI TRDLKDYKAT GPHVAVAKRL AARGVKIRPG TVISYIVLKG SGRIGDRAIP
721
FDEFDPTKHK YDAEYYIENQ VLPAVERILR AFGYRKEDLR YQKTRQVGLS AWLKPKGT
Sequence 27 (SEQ ID NO: 27)
Amino acid sequence of chimeric DNA polymerase Pod-II.
1
MASAILDVDY ITEEGKPVIR LFKKENGKFK IEHDRTFRPY IYALLRDDSK IEEVKKITGE
61
RHGKIVRIVD VEKVEKKFLG KPITVWKLYL EHPQDVPTIR EKVREHPAVV DIFEYDIPFA
121
KRYLIDKGLI PMEGEEELKI LAFDIETLYH EGEEFGKGPI IMISYADENE AKVITWKNID
181
LPYVEVVSSE REMIKRFLRI IREKDPDIIV TYNGDSFDFP YLAKRAEKLG IKLTIGRDGS
241
EPKMQRIGDM TAVEVKGRIH FDLYHVITRT INLPTYTLEA VYEAIFGKPK EKVYADEIAK
301
AWESGENLER VARYSMEDAK VTYELGKEFL PMEAQLSRLI GQSLWDVSRS STGNLVEWFL
361
LRKAYERNEL APNKPDEKEL ARRRQSYEGG YVKEPERGLW ENIVYLDFRS LYPSIIITHN
421
VSPDTLNREG CKEYDVAPQV GHRFCKDFPG FIPSLLGDLL EERQKIKKKM KATIDPIERK
481
LLDYRQRAIK ILANSYYGYY GYARARWYCK ECAESVTAWG REYITMTIKE IEEKYGFKVI
541
YSDTDGFFAT IPGADAETVK KKAMEFLKYI NAKLPGALEL EYEGFYKRGF FVTKKKYAVI
601
DEEGKITTRG LEIVRRDWSE IAKETQARVL ETILKHGDVE EAVRIVKEVI QKLANYEIPP
661
EKLAIYEQIT RPLHEYKAIG PHVAVAKKLA AKGVKIKPGM VIGYIVLRGD GPISNRAILA
721
EEYDPKKHKY DAEYYIENQV LPAVLRILEG FGYRKEDLRY QKTRQVGLTS WLNIKKS
Sequence 28 (SEQ ID NO: 28)
Amino acid sequence of chimeric DNA polymerase Kofu-III.
1
MASAILDTDY ITEDGKPVIR IFKKENGEFK IEYDRTFEPY FYALLKDDSA IEEVKKITAE
61
RHGTVVTVKR VEKVQKKFLG RPVEVWKLYF THPQDVPAIR DKIREHPAVI DIYEYDIPFA
121
KRYLIDKGLV PMEGDEELKM LAFDIETLYH EGEEFAEGPI LMISYADEEG ARVITWKNVD
181
LPYVDVVSTE REMIKRFLRV VKEKDPDVLI TYNGDNFDFA YLKKRCEKLG INFALGRDGS
241
EPKIQRMGDR FAVEVKGRIH FDLYPVIRRT INLPTYTLEA VYEAVFGQPK EKVYAEEITT
301
AWETGENLER VARYSMEDAK VTYELGKEFL PMEAQLSRLI GQSLWDVSRS STGNLVEWFL
361
LRKAYERNEL APNKPDEKEL ARRRQSYEGG YVKEPEKGLW ENIVYLDFRA LYPSIIITHN
421
VSPDTLNLEG CKNYDIAPQV GHKFCKDIPG FIPSLLGHLL EERQKIKTKM KETQDPIEKI
481
LLDYRQKAIK LLANSFYGYY GYAKARWYCK ECAESVTAWG RKYIELVWKE LEEKFGFKVL
541
YIDTDGLYAT IPGGESEEIK KKALEFLKYI NAKLPGALEL EYEGFYKRGF FVTKKKYAVI
601
DEEGKITTRG LEIVRRDWSE IAKETQARVL EALLKDGDVE KAVRIVKEVT EKLSKYEVPP
661
EKLVIHEQIT RDLKDYKATG PHVAVAKRLA ARGVKIRPGT VISYIVLKGS GRIGDRAIPF
721
DEFDPTKHKY DAEYYIENQV LPAVERILRA FGYRKEDLRY QKTRQVGLSA WLKPKGT
Sequence 29 (SEQ ID NO: 29)
Amino acid sequence of chimeric DNA polymerase Pod-III.
1
MASAILDVDY ITEEGKPVIR LFKKENGKFK IEHDRTFRPY IYALLRDDSK IEEVKKITGE
61
RHGKIVRIVD VEKVEKKFLG KPITVWKLYL EHPQDVPTIR EKVREHPAVV DIFEYDIPFA
121
KRYLIDKGLI PMEGEEELKI LAFDIETLYH EGEEFGKGPI IMISYADENE AKVITWKNID
181
LPYVEVVSSE REMIKRFLRI IREKDPDIIV TYNGDSFDFP YLAKRAEKLG IKLTIGRDGS
241
EPKMQRIGDM TAVEVKGRIH FDLYHVITRT INLPTYTLEA VYEAIFGKPK EKVYADEIAK
301
AWESGENLER VAKYSMEDAK ATYELGKEFL PMEIQLSRLV GQPLWDVSRS STGNLVEWFL
361
LRKAYERNEV APNKPSEEEY QRRLRESYTG GFVKEPERGL WENIVYLDFR SLYPSIIITH
421
NVSPDTLNRE GCKEYDVAPQ VGHRFCKDFP GFIPSLLGDL LEERQKIKKK MKATIDPIER
481
KLLDYRQRAI KILANSYYGY YGYARARWYC KECAESVTAW GREYITMTIK EIEEKYGFKV
541
IYSDTDGFFA TIPGADAETV KKKAMEFVKY INSKLPGLLE LEYEGFYKRG FFVTKKRYAV
601
IDEEGKVITR GLEIVRRDWS EIAKETQARV LETILKHGDV EEAVRIVKEV IQKLANYEIP
661
PEKLAIYEQI TRPLHEYKAI GPHVAVAKKL AAKGVKIKPG MVIGYIVLRG DGPISNRAIL
721
AEEYDPKKHK YDAEYYIENQ VLPAVLRILE GFGYRKEDLR YQKTRQVGLT SWLNIKKS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims. The articles “a”, “an”, and “the” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth herein. It should also be understood that any embodiment of the invention, e.g., any embodiment found within the prior art, can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the invention includes embodiments in which the order is so limited. Furthermore, where the claims recite a composition, the invention encompasses methods of using the composition and methods of making the composition. Where the claims recite a composition, it should be understood that the invention encompasses methods of using the composition and methods of making the composition.
All publications and patent documents cited in this application are incorporated by reference in their entirety to the same extent as if the contents of each individual publication or patent document were incorporated herein.
Faurholm, Bjarne, McEwan, Paul, Bourn, William, Rush, Gavin
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| Nov 03 2009 | Kapa Biosystems | (assignment on the face of the patent) | / | |||
| Oct 20 2011 | FAURHOLM, BJARNE | KAPABIOSYSTEMS | ASSIGNMENT OF ASSIGNORS INTEREST SEE DOCUMENT FOR DETAILS | 027116 | /0273 | |
| Oct 20 2011 | MCEWAN, PAUL | KAPABIOSYSTEMS | ASSIGNMENT OF ASSIGNORS INTEREST SEE DOCUMENT FOR DETAILS | 027116 | /0273 | |
| Oct 20 2011 | BOURN, WILLIAM | KAPABIOSYSTEMS | ASSIGNMENT OF ASSIGNORS INTEREST SEE DOCUMENT FOR DETAILS | 027116 | /0273 | |
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